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UNIVERSIDADE ESTADUAL DE SANTA CRUZ
PRÓ-REITORIA DE PESQUISA E PÓS-GRADUAÇÃO
Programa de Pós-graduação em Genética e Biologia Molecular
FABIANA APARECIDA CAVALCANTE SILVA
TcPR-10: Mecanismo de transporte e ação em fungos
ILHÉUS – BAHIA – BRASIL
Fevereiro – 2013
FABIANA APARECIDA CAVALCANTE SILVA
TcPR-10: Mecanismo de transporte e ação em fungos
Tese apresentada à Universidade
Estadual de Santa Cruz, como
parte das exigências para obtenção
do título de Doutor em Genética e
Biologia Molecular
Orientador: Prof. Dr. Abelmon da Silva Gesteira
Co-orientadores: Profa. Dra. Fabienne Micheli
Prof. Dr. Márcio Gilberto Costa
ILHÉUS – BAHIA – BRASIL
Fevereiro – 2013
S586 Silva, Fabiana Aparecida Cavalcante. TcPR-10: mecanismo de transporte e ação em Fungos / Fabiana Aparecida Cavalcante Silva. – Ilhéus, BA: UESC, 2013. xv, 93f. : il. Orientador: Abelmon da Silva Gesteira. Tese (doutorado) – Universidade Estadual de Santa Cruz. Programa de Pós-Graduação em Ge- nética e Biologia Molecular. Inclui referência bibliográfica.
1. Fungos fitopatogênicos – Controle biológico. 2. Saccharomyces cerevisiae. 3. Oxigênio – Trans- porte fisiológico. 4. Proteínas. 5. Vassoura-de-bruxa (Fitopatologia). 6. Plantas – Parasito. I. Título. CDD 632.4
II
FABIANA APARECIDA CAVALCANTE SILVA
TcPR-10: Mecanismo de transporte e ação em fungos
Tese apresentada à Universidade
Estadual de Santa Cruz, como parte das
exigências para obtenção do título de
Doutor em Genética e Biologia Molecular.
Ilhéus, 27 de Fevereiro de 2013
BANCA EXAMINADORA
___________________________ ___________________________
Drª. Cristina Caribé Drª Helena Costa
(DCB/UESC) (DCB/UESC)
___________________________ ___________________________
Drª Jane Lima dos Santos Drª. Juliana Freitas Ástua
(DCB/ UESC) (EMBRAPA)
_____________________________
Dr. Abelmon da Silva Gesteira
(EMBRAPA - Orientador)
II
"Un peu de science éloigne de Dieu, beaucoup de science y ramène."
"Um pouco de ciência nos afasta de Deus. Muito, nos aproxima."
Louis Pasteur, 1957
III
A meus pais, Francisco de Assis da Silva e Maria Aparecida C. Silva, que
nunca mediram esforços para que pudesse ter o que consideraram como o
melhor do conhecimento, inúmeras experiências de vida e ser o que sou hoje,
não posso somente agradecer, mas dedicar-lhes esse novo sonho realizado e
retribuir-lhes sempre o amor que têm por mim.
Aos meus irmãos Fábio, Fabíola, Fabilson e Fabianderson . Agradeço
imensamente pela força e apoio sempre presentes e pela alegria
da convivência.
DEDICO
IV
AGRADECIMENTOS
Á Deus, pela vida, saúde, discernimento, paciência, e por sua presença em
todas as etapas desta caminhada. Pelas experiências, bem sucedidas ou não,
mas sempre fonte de aprendizado. Pela minha família, professores, colegas de
trabalho e todos os amigos;
À Universidade Estadual de Santa Cruz, pela oportunidade de realização deste
curso;
A CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)
pela concessão da bolsa;
Ao professor Dr. Abelmon da Silva Gesteira pela orientação, paciência,
disponibilidade e preciosos ensinamentos. Agradeço a oportunidade de fazer
parte de seu grupo de pesquisa e sempre lembrarei do exemplo de ética,
profissionalismo, dedicação, doação e muito entusiasmo pelo trabalho;
Aos co-orientadores Márcio Gilberto Cardoso Costa e Fabienne Micheli pelo
apoio em todas as etapas de realização do trabalho.
A Dra. Cristina Pungartinik pelo imenso apoio e dedicação às discussões e
sugestões ao trabalho. Mesmo não sendo oficialmente co-orientadora, sua
confiança e amizade foram de grande importância para o bom desenvolvimento
desse trabalho.
Ao Dr. Martin Brendel pela colaboração e ensinamentos na elaboração do
trabalho envolvendo leveduras.
As grandes amigas: Ana Cácia, Camila, Luana Moreira, Sara Menezes,
Luciana Cidade, Ana Camila e Daniela Koop. Agradeço todo o apoio e amizade
em todos os momentos.
Aos amigos Thyago Cardoso, Gileno Lacerda, Sanderson Tarcizo, Ricardo
Porto e Carlos Ivan pela ajuda nos dias e noites de trabalho, conhecimentos
compartilhados, e força nos momentos difíceis.
Aos amigos-companheiros da Turma de Doutorado 2008.1: Jeiza, Cinthia,
Vania, Rogério, Samuel Saito, Ana Carolina Santini, Olivia e Heliana pela
amizade e companheirismo ao longo do curso
V
Aos amigos André Santiago, Juliano Mendes, Juliano Santana, Leila, Alanna. e
Dayane pelos ensinamentos, incentivos e colaborações.
Aos colegas do Laboratório de Biologia Molecular Dayane, Diana Matos,
Edson, Heliana e Leandro pelo companheirismo no laboratório e principalmente
pelos bons momentos que passamos.
Aos colegas do Laboratório de Cultura de Tecidos Vegetais Jamilly, Amanda,
Laís, Cristina Martins, Verônica, Antônio Carlos (Pelé) e Virgínia pela
disponibilidade, ensinamentos e excelente convivência ao longo dos anos.
Aos Dr. Marco Antônio Costa pela ajuda e disponibilização do Laboratório de
Citogenética e os colegas Rodolpho, Adriane Barth e Igor pela ajuda nos
experimentos de microscopia.
Aos colegas dos Laboratórios de Marcadores Moleculares, Imunologia, Biologia
de Fungos e Biotecnologia Microbiana, em especial Ronaldo, Gil, Samuel Saito
e Tatiana Basso.
Aos colegas do Lab. De Proteômica e Genômica, em especial Ana Camila,
Emanuele, Joise, Juliano e Luciana Camilo.
Aos amigos do CEPEC/CEPLAC por serem um refúgio e um apoio sempre que
foi necessário, em especial Kaleandra, Livia, Leila e Rogério.
Aos amigos de Recife por se fazerem presentes, mesmo que a distancia nos
separasse: Denise Bacelar Renata Rodrigues, Vladimir Silveira, Mariana,
Layana, Hi Meet Shiue, Ingrid, Oswaldo, Carolina Felinto. Obrigada por não me
deixarem abater.
A todas as minhas companheiras de republica que aguentaram todos os
momentos de estresse apoiando, mesmo sem entender muito, mas sempre
foram fundamentais: Lisiane, Lilian e Renally.
Aos funcionários da UESC: Dona Gilda e Fabricia.
Meus sinceros agradecimentos. Sem vocês não teria chegado até aqui.
VI
ÍNDICE
Pag.
LISTA DE FIGURAS IX
LISTA DE TABELAS XI
EXTRATO XII
ABSTRACT XIV
I. INTRODUÇÃO. 16
II. REVISÃO BIBLIOGRÁFICA 19
1. Theobroma cacao 19
2. Moniliophthora perniciosa 21
3. Interação planta – patógeno: Estudos moleculares 24
3.1. Patossistema T. cacao x M. perniciosa 26
4. Proteínas relacionadas à Patogênese (PR proteínas) 27
4.1. Proteínas relacionadas à Patogênese da família 10 (PR-10) 29
5. Ferramentas proteômicas na interação planta X patógeno 30
6. Saccharomyces cerevisiae: Transporte ABC, autofagia e formação
de vacúolos 31
III. REFERENCIAS BIBLIOGRÁFICAS 38
IV. CAPÍTULO 1 44
V. CAPÍTULO 2 64
VI. CONCLUSÕES GERAIS 93
VII
LISTA DE FIGURAS
Revisão Bibliográfica
Figura 1. Produção mundial de amêndoas de cacau no ano de 2011 nos
principais países produtores. FAO: 30/01/2013
Figura 2. Ciclo de vida do fungo M. perniciosa.
Figura 3. Resumo esquemático dos processos metabólicos envolvidos no
mecanismo de defesa das plantas ao ataque de patógenos
Figura 4. Representação esquemática das etapas relacionadas as rotas
autofágicas.
Figura 5. Transporte de proteínas para o vacúolo em leveduras
Capítulo 1
Figure 1. Protein profile in 2-DE gel Moniliophthora perniciosa fungus treated
with the antifungal protein TcPR-10 obtained from cacao. Treatments: A-
Control (no TcPR-10); B- 30 min; C- 60 min and D- 120 min after exposure to
TcPR-10. The gels were stained with Coomassie Coloidal Blue G-250 solution.
Line MW: molecular weight marker proteins (kDa). → Excision place gel to
differentially expressed proteins identificated by mass spectrometry.
Figure 2. Representations of the distribution of identified differentially
expressed proteins identified M perniciosa according to their biological process.
Categorizations were based on information provided by the online resource
UniProt classification system.
Capítulo 2
Figure 1. Survival of S. cerevisiae in exponential growth phase exposed to
TcPR-10p (3 µg/mL) for 0, 1, 6 and 24 h: A) BY10000 (); pdr5Δ (X); pdr10Δ
(); pdr12Δ (); pdr15Δ () and yor1Δ (▲); B) BY10000 (), pdr11Δ() and
atg8Δ ();
Figure 2. WT cells and mutant snq2Δ of S. cerevisiae in exponential phase
observed in real time microscope. A) control: WT and snq2Δ kept for 2 and 4 h;
B) cells exposed to TcPR-10p (3 µg/mL): WT for 0, 60 and 90 min and snq2Δ
for 0 , 90 and 120 min. Bar: 10 µm.
VIII
Figure 3. Observation of vacuoles in S. cerevisiae in exponential growth phase
exposed to TcPR-10p (3 µg/mL) 0 and 6 h: A) BY10000, B) snq2Δ, C) BY10000
and D) pdr11Δ. Bar: 50 µm.
Figure 4. Survival of yeast transformed with single-copy plasmid containing
MpATG8 exposed to TcPR-10p (3 µg/mL) for 0, 12, 24 and 48 h. AP01 ( );
AP02 (); AP03 () and AP04 ().
Figure 5. Production of ROS observed in epifluorescence photomicroscopy
after 0 and 48 h exposure to TcPR-10p (3 µg/mL). A) AP01, B) AP02, C) AP03
and D) AP04. Bar: 50 µm.
Figure 6. Hyphae of M. perniciosa observed in real time microscope. A)
Control; B) Hyphae exposed to TcPR-10 (3 µg/mL)
SUPPLEMENTARY MATERIAL
Figures:
Supp 1. Nucleotide and amino acid sequences of Moniliophthora perniciosa
autophagy-related protein 8 precursor (gi|189380199|and gi|189380200).
Asterisk represents open reading frame termination codon.
Supp 2. Molecular cloning of gene MpAtg8 into vector pRS313. E= EcoR I; H=
HIS3; A= ARSH4; R= ampR.
Supp 3. – PCR of colony confirming insertion of the MpATG8 gene, C+
represents the positive control, in which DNA of M. pernicious was used. C+
represents the positive control, in which DNA of M. pernicious was used.
Movies:
Supp. 4 – WT 1h TcPR-10. mov: LOG phase cells of S. cerevisiae WT
observed in real time microscopy after exposure to TcPR-10p (3µg/mL) for 1h
Supp. 5 – MPTcPR-10. mov: M. perniciosa hyphae observed in real time
microscopy after exposure to TcPR-10p (3 µg/mL) for 1h.
IX
LISTA DE TABELAS
Revisão Bibliográfica
Tabela 1. Famílias de proteínas relacionadas à patogênese (PR).
Tabela 2. Descrição de bombas de efluxo de drogas encontradas em S. cerevisiae
Capítulo 1.
Table 1. Distribution of spots between treatments (Control, 30 min, 60 min and 120
min) and between combinations of these treatments.
Capítulo 2
Table 1: Yeast strains and plasmid
X
EXTRATO
SILVA, Fabiana Aparecida Cavalcante Silva, Msc, Universidade Estadual
de Santa Cruz, Ilhéus, Fevereiro de 2013. TcPR-10: Mecanismo de
transporte e ação em fungos. Orientador: Abelmon da Silva Gesteira. Co-
orientador: Márcio Gilberto Costa.
A vassoura-de-bruxa, causada pelo fungo hemibiotrófico Moniliophthora perniciosa (Stahel), é a principal causa do declínio econômico da cultura cacaueira na região Sul da Bahia. Desta forma diversos métodos para o controle ou melhor entendimento desta doença tem sido desenvolvidos através de ferramentas genômicas e proteômicas. A análise funcional de genes que codificam para proteínas PR (Pathogenesis related proteins) são de grande importância principalmente por serem expressas sob condições de patogênese ou pressões ambientais. A partir de uma biblioteca de cDNA da interação entre Theobroma cacao e M. perniciosa, foi identificada a proteína TcPR-10, cujos trabalhos posteriores revelaram forte ação fungicida e de ribonuclease contra M. perniciosa e Saccharomyces cerevisiae. Mutantes de S. cerevisiae para genes de reparação do DNA, transporte de membrana, transporte de metais e defesas antioxidantes foram expostos a TcPR10 indicando um possível transporte ativo desta proteína em células de levedura em fase logarítmica de reprodução (LOG) e sua atividade antifúngica parece estar associada à transportadores de membrana e à ação de permeases. Diante destes resultados prévios houve a necessidade de identificar quais proteínas o fungo causador da vassoura-de-bruxa expressa quando exposto a TcPR-10 e quais mecanismos de ação e transporte TcPR-10 utiliza para penetração na célula fungica. A análise proteômica foi utilizada com o objetivo de identificar proteínas diferencialmente expressas em M. perniciosa em resposta à TcPR-10. As hifas do fungo foram expostas a proteína heteróloga TcPR-10 (3 µg/mL) em quatro tratamentos: controle (sem TcPR-10), 30min, 60min e 120 min após exposição a proteína antifúngica. Os mapas bidimensionais apresentaram 191 proteínas diferencialmente expressas, das quais 55 foram identificadas por espectrometria de massas. As proteínas identificadas foram relacionadas ao metabolismo celular, resposta ao estresse, a ligação de zinco, mecanismo de fosforilação, transporte, autofagia, reparo do DNA e oxidoredutases. Destas 29% se referiam a proteínas de resposta ao estresse e 25% a oxidoredutases (25%) principalmente nos tratamentos controle e 30 min, reduzindo sua expressão aos 120 min, Os estresse oxidativo causado por TcPR-10 explica o aumento da expressão destas duas classes, resposta ao estresse e oxiduredutase, que atuam no processo de reparação de danos. Além desta foram identificadas proteínas de detoxificação (autofagia) e esterol importantes para manutenção da homeostase celular em fungos. Sabendo o efeito que TcPR-10 causa em M. perniciosa é necessário entender qual o mecanismo de transporte para o interior da célula são utlizados e quais efeitos causam. Desta forma foram utilizados mutantes de S. cerevisiae para transportadores de
XI
membrana do tipo ABC (ATP-binding cassete), autofagia e ligados a formação de vacúolo, além da visualização via microscopia em tempo real de células de S. cerevisiae e M. perniciosa tratadas com TcPR-10. O mutante isogênico pdr11Δ apresentou maior resistência a TcPR-10 quando comparado com a linhagem selvagem (WT), de forma contrária, atg8Δ, mutante deficiente em autofagia, apresentou 10 vezes menor sensibilidade. O corante vermelho neutro, ideal para coloração de vacúolos, demonstrou que os mutantes pdr11Δ e snq2Δ foram mais resistentes a TcPR-10 quando comparados a WT devido a uma redução da formação de vacúolos. O mesmo foi observado com a microscopia em tempo real com uma menor formação de vacúolos nos mutantes snq2Δ em relação a WT, e em hifas tratadas de M. perniciosa. O mecanismo autofágico foi testado através da expressão heteróloga de quatro linhagens isogênicas de S. cerevisiae: selvagem contendo uma cópia do gene ATG8 de M. perniciosa (MpATG8), selvagem, atg8Δ (mutante) contendo (MpATG8) e atg8Δ. Com os resultados obtidos podemos sugerir que a proteína TcPR-10 utiliza a via de penetração celular similar à dos esteróis, tal como sugerido pela resistência de mutantes pdr11Δ snq2Δ e que o acúmulo observado nos vacúolos podem ser devido a atividade intracelular de TcPR-10 em leveduras e M. perniciosa, além disso a rota autofágica é essencial para a resistência da linhagem selvagem à TcPR-10 eliminando os danos causados pelo estresse oxidativo. As proteínas de M. perniciosa, responsivas a TcPR-10, identificadas por espectrometria de massas estão incluídas em várias rotas bioquímicas sugerindo prováveis modos de ação de agentes antifúngicos assim como possíveis mecanismos de transporte poderão ser considerados a partir dos resultados obtidos.
Palavras- chave: Proteína relacionada a patogênese 10 (PR-10), Espécies
reativas a oxigenio (ROS), proteínas diferencialmente expressas,
oxidoredutases Transportadores de membrana ABC, Autofagia.
XII
ABSTRACT
SILVA, Fabiana Aparecida Cavalcante Silva, Msc, Universidade Estadual
de Santa Cruz, Ilhéus, February of 2013. TcPR-10: Transport and mode of
action. Advisor: Abelmon da Silva Gesteira. Advisor Committee: Márcio
Gilberto Costa.
The witches' broom, caused by the hemibiotrophic fungus Moniliophthora perniciosa (Stahel) is the main cause of the economic decline of the cocoa crop in Southern Bahia. Thus a search for methods of disease control has been developed through genomic and proteomic studies. Functional analysis of genes encoding PR (Pathogenesis Related) proteins are important primarily when expressed under conditions of environmental stress or pathogenesis. From a cDNA library of the interaction between Theobroma cacao and M. perniciosa we identified TcPR-10, later show to have strong fungicidal and ribonuclease action against M. perniciosa and Saccharomyces cerevisiae. Mutants of S. cerevisiae genes for DNA repair, membrane transport, transport of metals and antioxidant defenses were exposed to TcPR10 indicating a possible active transport of this protein in yeast cells in logarithmic phase of reproduction (LOG) and their antifungal activity seems to be associated with the membrane transport and permeases action. Given these previous results it was necessary to identify which proteins of M. perniciosa were expressed when exposed to TcPR-10 and which mechanisms of action and transport of TcPR-10 are made to penetrate the fungal cell. A proteomic analysis was used in order to identify proteins differentially expressed in M. perniciosa in response to TcPR-10. The hyphae of the fungus were exposed to TcPR-10 heterologous protein (3 mg/mL) in four treatments: Control (no TcPR-10), 30min, 60min and 120min after exposure to antifungal protein. The two-dimensional maps showed 191 differentially expressed proteins 55 of which were identified by mass spectrometry. The proteins were related to cell metabolism, stress response, zinc binding mechanism, phosphorylation, transport, autophagy, DNA repair and oxidoreductases. Of these 29% refered to proteins and stress response 25% were oxidoreductases mainly in control treatments and 30 min, with expression at 120 min. The oxidative stress caused by TcPR-10 explains the increased expression of these two classes, stress response and oxiduredutase, which act in the process of damage repair. In addition detoxification proteins were identified (autophagy) and sterol important for maintenance of cellular homeostasis in fungi. Knowing the effect that causes TcPR-10 in M. perniciosa is necessary to understand the mechanism of transport to the cell interior and effects that causes. Thus we used mutants of S. cerevisiae for type membrane transporters ABC (ATP-binding cassette), and autophagic vacuole formation attached to, and visualization via microscopy in real time cell S. cerevisiae and M. perniciosa treated with TcPR-10. The isogenic mutant was more resistant to pdr11Δ TcPR-10 when compared to the wild type (WT), contrary, atg8Δ, mutant
XIII
deficient in autophagy, showed 10 times lower sensitivity. The neutral red dye, suitable, showed that the mutants were snq2Δ and pdr11Δ, more resistant to TcPR-10 when compared to WT due to a reduction in the formation of vacuoles. The same was observed with microscopy in real time with a lower formation of vacuoles in the mutants compared to WT and snq2Δ, and treated hyphae of M. perniciosa. The autophagic mechanism was tested by heterologous expression of four strains of S. cerevisiae: wild containing one copy of the gene ATG8 of M. perniciosa (MpATG8), WT, atg8Δ (mutant) containing (MpATG8) and atg8Δ. This results may suggest that the protein TcPR-10 uses the route of cell penetration similar to the sterols, as suggested by the resistance of mutants pdr11Δ snq2Δ and the observed accumulation in the vacuoles may be due to intracellular activity of TcPR-10 in yeast and M. perniciosa also the autophagic route is essential for the resistance of the wild strain TcPR-10 eliminating the damage caused by oxidative stress. The proteins of M. perniciosa, responsive TcPR-10, identified by mass spectrometry are included in several biochemical pathways suggest possible modes of action of antifungal agents as well as possible mechanisms of transport.
Key words: Pathogenesis related protein family 10 (PR-10), reactive oxygen
species (ROS), differentially expressed proteins, oxidureductases ATP-binding
cassette (ABC) family, Autophagy
14
I. INTRODUÇÃO
O cacau (Theobroma cacao) é uma das culturas mais importantes do
mundo, com uma produção de mais de 4 milhões de toneladas no ano de 2011
(FAOSTAT, 2013), sendo cultivado em aproximadamente 50 países (KNIGHT,
2000). Na região Sul da Bahia o cultivo do cacau, além do caráter econômico,
tem uma responsabilidade ecológica visto que esta cultura está associada à
preservação da mata atlântica nativa em virtude do modelo de cultivo, feito sob
a mata primária em um sistema denominado cabruca (SAMBUICHI et al.,
2012). Além da sua principal aplicação como base para fabricação de
chocolate, o cacau é matéria prima de subprodutos aplicados na indústria de
cosméticos, bebidas, geleias, sorvetes, sucos, dentre outros (ALMEIDA-
VALLE, 2007).
A introdução do fungo hemibiotrófico Moniliophthora (= Crinipellis)
perniciosa (AIME e PHILLIPS-MORA, 2005), causador da vassoura de bruxa,
tem causado sérios prejuízos socioeconômicos ao nível mundial e,
regionalmente, foi causa do êxodo rural que acarretou na favelização dos
grandes centros urbanos do estado da Bahia. Diante do declínio da lavoura
cacaueira frente à vassoura-de-bruxa, o entendimento dos mecanismos
envolvidos no patossistema T. cacau x M. perniciosa é de grande importância
na tentativa de alcançar um controle efetivo da doença. Desta forma vários
estudos genômicos, proteômicos e funcionais vêm sendo desenvolvidos.
O projeto Genoma do fungo Moniliophthora perniciosa
(http://www.lge.ibi.unicamp.br/vassoura/) envolveu várias instituições públicas
brasileiras (EMBRAPA Cenargen, Ceplac, UESC, Unicamp, UEFS, UFBA) no
ano 2000. Em seguida, no ano de 2004, foi dado inicio ao Projeto Proteoma do
fungo (Rede Nacional de Proteômica), visando caracterizar bioquimicamente o
fungo e a planta. Gesteira et al. (2007) através de bibliotecas de cDNA da
interação entre o fungo e cultivares resistentes e suscetíveis de cacau,
identificaram sequências relacionadas a diversos aspectos da interação, como
inibidores de proteases, inibidores de tripsina (Its), cistatinas, proteínas
relacionadas a patogênese (TcPR-10) e indutores de necrose (NEP).
15
As proteínas relacionadas à patogênese (Proteínas PR) são expressas
em mono e dicotiledôneas sob condições de patogênese ou pressões
ambientais e, em alguns casos, possuem um papel no desenvolvimento geral
da planta. As proteínas PR são classificadas em 17 famílias de acordo com a
massa molecular, ponto isoelétrico, localização e atividade biológica (LIU e
EKRAMODDOULLAH, 2006; VAN LOON, REP e PIETERSE, 2006). As
proteínas da família 10 (PR-10) apresentam três sítios comuns de fosforilação
sugerindo uma função como RNase geral ou como RNase específica contra
RNA exógeno (GRAHAM et al., 2003; PARK et al., 2004; KIM et al., 2008).
Trabalhos recentes identificaram atividade DNAse das proteínas PR10 em
arroz (KIM et al., 2011) e uva (HE et al., 2012) sugerindo uma ação de
nuclease no processo de morte celular programada.
A proteína TcPR-10 foi caracterizada por Pungartnik et al. (2009a) e
Menezes et al. (2012) que observaram atividade de ribonuclease e antifúngica
da proteína heteróloga contra M. perniciosa e Saccharomyces cerevisiae.
TcPR-10 foi colocada em contato com mutantes de S. cerevisiae para genes de
reparação do DNA, transporte de membrana, transporte de metais e defesas.
Os resultados indicaram que a atividade antifúngica da proteína podem estar
associados a transportadores de membrana e à ação de permeases e que
TcPR-10 parece utilizar um transporte ativo em células de levedura em fase
logarítmica de reprodução (LOG).
O fungo M. perniciosa tem sido fonte de estudos que buscam
caracterizar aspectos fisiológicos como o processo de germinação dos esporos
(MARES, 2012), regulação de quitinase (LOPES et al., 2008) e análise do
secretoma durante o processo de infecção e colonização do hospedeiro
(ALVIM et al., 2009), dentre outros. O uso da analise proteômica para
identificação de proteínas diferencialmente expressas tem sido amplamente
utilizada para compreensão dos mecanismos utilizados pelo patógeno na
interação com o hospedeiro ou em resposta a drogas. Ebanks et al. (2006)
compararam perfis protéicos em leveduras e hifas de Candida albicans
buscando identificar proteínas da parede celular e associadas à parede,
utilizando as técnicas de MudPit e géis bidimensionais. Este trabalho resultou
16
na identificação de 29 proteínas das quais 17 foram identificadas apenas em
hifas, quatro em levedura, e oito foram identificados na levedura e hifas. As
proteínas expressas pelo fungo causador da vassoura-de-bruxa na presença
de antifúngicos ainda são pouco conhecidas, principalmente utilizando
ferramentas proteômicas. Este trabalho apresenta o primeiro mapa protéico de
M. perniciosa quando exposto a uma proteína antifúngica isolada do cacau
(TcPR-10) e busca compreender as respostas bioquímicas relevantes no
mecanismo de patogenicidade deste fungo.
Considerando o mecanismo de resposta de M. perniciosa quando
exposta à TcPR-10, outro importante aspecto da ação desta proteína
antifúngica que necessita ser melhor elucidado é seu mecanismo de
penetração no fungo, in vivo, e as consequências decorrentes desta
penetração. Neste contexto a utilização de mutantes de S. cerevisiae para
transportadores de membrana do tipo ABC, formação de vacúolo e autofagia
são ferramentas úteis para a resolução destes questionamentos.
Estudos funcional e bioquímico de genes de interesse permitem inferir
possíveis vias metabólicas envolvidas na interação entre patógeno e seu
hospedeiro, bem como desenvolver estratégias mais eficientes de controle de
fitopatógenos. Desta forma este trabalho teve como objetivo avaliar o
mecanismo de transporte e aspectos de ação da proteína TcPR-10 em S.
cerevisiae e M. perniciosa utilizando análises in vitro e ferramentas
proteômicas.
17
II. REVISÃO BIBLIOGRÁFICA
1. Theobroma cacao
O cacaueiro (Theobroma cacao L.) é uma planta pertencente à família
Malvaceae, arbórea, eudicotiledônea, típica de clima tropical úmido que vegeta
no sub-bosque e compreende plantas preferencialmente alógamas (SILVA et
al., 2001). É cultivado e reproduzido por sementes podendo apresentar altura
entre 5 e 10 m, havendo registros de indivíduos com 50 a 75 m (ALVERSON et
al., 1999), Os representantes desta espécie são diploides (2n= 2x= 20), com
genoma considerado pequeno (411 – 494 Mb) (FIGUEIRA, JANICK e
GOLDSBROUGH, 1992; LANAUD, HAMON e DUPERRAY, 1992; ARGOUT et
al., 2011),
A espécie T. cacao possui como centro de origem, provavelmente, as
nascentes do rio Amazonas e Orinoco e atualmente está distribuído nas
florestas tropicais da América Central, América do Sul, Ásia e África
(CHEESMAN, 1944; COE, COE e HUXTABLE, 1996; MARITA et al., 2001). O
cacaueiro pode ser subdividido morfogeograficamente em: i. Crioulo (T.cacao
ssp. cacao) oriundos da América Central e México; ii. Forasteiro (T. cacao ssp.
sphaeorocarpum) oriundo da América do Sul; e iii. Trinitario, uma espécie
híbrida do cruzamento entre os dois tipos anteriormente citados (MOTAMAYOR
et al., 2002).
O cacau é uma commodity internacionalmente reconhecida
principalmente por ser a matéria prima na produção do chocolate, um produto
amplamente consumido devido ao seu valor energético e nutritivo
(MOTAMAYOR et al., 2008). Além disso, possui outros importantes mercados
como: manteiga de cacau, utilizada na indústria farmacêutica e cosmética, e a
polpa: utilizada para fabricação de sorvetes, sucos, licores, geleias, vinho,
vinagre, inclusive no mercado internacional (ALMEIDA e VALLE, 2007).
Atualmente o Brasil é o sexto produtor mundial de cacau tendo
produzido 4,32 milhões de toneladas de amêndoas em 2011 (Figura 1). Apesar
do histórico de exportação alcançado pela indústria cacaueira brasileira,
atualmente a produção é utilizada para utilização local. Este declínio se deu a
18
partir de 1987 quando o setor cacaueiro sofreu grave crise causada pelo baixo
preço atribuído ao produto, o monocultivo, a introdução da doença vassoura de
bruxa na região sul da Bahia, além da baixa competitividade do setor, que
exige custos relativamente altos e a exploração de áreas de cultivo antigas e
pouco produtivas, submetidas a um manejo inadequado (SOUZA, DIAS e
DIAS, 2001; MEINHARDT et al., 2008).
Figura 1. Produção mundial de amêndoas de cacau no ano de 2011 nos principais
países produtores. FAO: 30/01/2013
19
Dentre as causas que acarretam a queda na produtividade do cacau
destaca-se o ataque de patógenos que se estabelecem em função das
condições climáticas da área na qual a planta esta inserida. Os principais
patógenos do cacaueiro são diversas espécies de Phytophthora, além de
Moniliophthora roreri e Moniliophthora perniciosa (ALLEGRE et al., 2012). No
Brasil o fungo M. perniciosa, causador da doença vassoura-de-bruxa, destaca-
se como principalmente patógeno da cultura do cacau.
2. Moniliophthora perniciosa:
O fungo Moniliophthora perniciosa (Stahel), causador da doença do
cacaueiro vassoura-de-bruxa, pertence à classe dos Basidiomycetes, ordem
Agaricales, família Marasmiaceae (AIME e PHILLIPS-MORA, 2005). A
propagação deste fungo ocorre com a liberação de esporos, os basidiósporos,
a partir de basidiocarpos. Os basidiósporos são capazes de infectar qualquer
tecido meristemático e são considerados os únicos propágulos infectivos
descritos para este patógeno. O processo infectivo pode ocorrer também
através da abertura dos estômatos pelos tubos germinativos dos basidiósporos
e pelas inflorescências não enrijecidas (EVANS, 1980; FRIAS, PURDY e
SCHMIDT, 1991).
O fungo apresenta ciclo de vida hemibiotrófico, desta forma o
desencadeamento da doença apresenta duas fases distintas: a biotrófica e a
necrotrófica ou saprofítica. Na fase biotrófica, quando a densidade do micélio é
baixa se apresentando na forma monocariótica intercelular, inicia-se o processo
de hipertrofia e hiperplasia dos tecidos, perda de dominância apical e
proliferação de meristemas axilares, resultando na formação de um ramo
anormal, conhecido como vassoura verde. A infecção nas flores resulta na
formação de pequenos frutos partenocárpicos ou vassoura de almofada. A fase
necrotrófica ou saprofítica tem início entre 1 a 2 meses do desenvolvimento da
doença, quando o fungo assume sua forma dicariótica intracelular, causando
necrose e morte dos tecidos infectados, formando ramos denominados
vassoura seca (EVANS, 1980; PENMAN et al., 2000; MEINHARDT et al.,
2008). As principais fontes de produção de basidiocarpos são as vassouras
20
descobertas e caídas dentro do cacaueiro, vassouras ou almofadas florais e
frutos doentes e vassouras necróticas vegetativas na copa do cacaueiro
(PURDY e SCHMIDT, 1996; COSTA et al., 1997). O ciclo da doença se
completa com a formação do basidiocarpo seguida da formação dos esporos
em qualquer tecido necrótico infectado (SCARPARI et al., 2005) (Figura 2).
A vassoura-de-bruxa foi introduzida na região sul da Bahia em 1989,
causando prejuízos econômicos e socioambientais. Pires et al. (1999)
relataram uma queda de 15% para 4,3% na produção de cacau entre os
períodos de 1989 e 1998. Este declínio acentuado desde então deve-se
principalmente a elevada susceptibilidade de algumas variedades de cacaueiro
e a grande severidade do patógeno associados ao clima favorável da região
resultando em fatores adequados à disseminação da doença (LUZ et al., 1997).
Diante das perdas causadas pela vassoura-de-bruxa diversas ações no
âmbito econômico e científico foram iniciadas buscando estabelecer um
controle efetivo da doença. Estudos têm sido desenvolvidos com o intuito de
melhor compreender a mecanismo de ação de M. perniciosa e assim permitir o
desenvolvimento de ferramentas de controle da vassoura-de-bruxa. Destacam-
se os trabalhos desenvolvidos pela UESC (Universidade Estadual de Santa
Cruz), CEPLAC (Comissão Executiva do Plano da Lavoura do Cacaueiro) e
CEPEC (Centro de Pesquisas do Cacau) que atuam em diversas áreas de
pesquisas voltadas ao controle da doença; além do projeto Renobruxa – Rede
do Renorbio – Vassoura-de-Bruxa cujo objetivo é dar suporte tecnológico para
revitalização da cacauicultura baiana e nacional a partir do controle do fungo
(FILHO, 2010).
21
Figura 2. Ciclo de vida do fungo M. perniciosa. Figura 1: Ciclo de
vida de M. perniciosa; A) o basidiocarpo é a estrutura reprodutiva
que contém os basidiósporos , B) os basidiósporos podem penetrar
no tecido de forma direta ou pela abertura dos estômatos, C) após a
penetração fungica o micélio monocariótico é intercelular, D) a
presença do pat ógeno gera hiperplasia nos órgãos infectados
ocasionando a denominada “vassoura verde”, E) sobre condições
apropriadas o micélio passa a ser dicariótico e se localiza
intracelularmente, F) a destruição celular gera a necrose e
consequente morte do tecido infectado, caracterizando a “vassoura
seca”, G) o micélio presente se diferencia na estrutura reprodutiva
(basidiocarpo) que contém novas unidades infectivas (basidiósporos)
iniciando o ciclo. O fundo cinza representa as etapas da fase
biotrófica, em azul fase saprofítica. Adaptado de Ceita (2007)
(Adaptada de Ceita et al., 2007).
22
3. Interação planta – patógeno: Estudos moleculares
As plantas são continuamente expostas a estresses bióticos e abióticos
ao longo do seu ciclo de vida desenvolvendo mecanismos de resposta a tais
eventos. Dentre os agentes causadores de estresse biótico encontram-se os
microrganismos que, para serem patogênicos, devem ser capazes de invadir a
planta penetrando diretamente através da superfície de folhas e raízes ou
entrando através de aberturas como estômatos ou ferimentos. A doença
provocada por esta invasão causa desequilíbrio nas plantas como o desvio de
nutrientes e metabólitos secundários, produção de toxinas nocivas às plantas
e, muitas vezes, a morte (CHISHOLM et al., 2006; CHIVASA et al., 2006).
A interação existente entre plantas e micro-organismos é de grande
interesse econômico uma vez que pode levar a grandes perdas na
produtividade da cultura, desta forma o entendimento dos mecanismos
bioquímicos de defesa são de grande importância do ponto de vista
agronômico.
O reconhecimento de um patógeno pela planta desencadeia várias
reações bioquímicas de defesa que levam à produção de Espécies Ativas de
Oxigênio’ (EAO’s) ou Reactive Oxygen Intermediates (ROI) ou ainda Reactive
Oxygen Species (ROS) (TORRES, JONES e DANGL, 2006). Em seguida
ocorre a resposta hipersensitiva (RH) com morte celular programada (MCP), e
resposta sistêmica adquirida (RSA ou SAR- Systemic acquired resistance)
(GOZZO, 2003) (Figura 3).
A resposta hipersensitiva ou de hipersensibilidade inclui o mecanismo de
morte celular programada localizada nas células situadas ao redor do ponto de
infecção (MUR et al., 2008). Estudos recentes indicam que HR está
diretamente associada uma resposta rápida e robusta que induz a produção de
compostos secundários antimicrobianos, ativação transcricional de uma série
de genes que codificam para enzimas líticas (quitinases, glucanases e
proteases) e proteínas antimicrobianas (defensinas) (GASSMANN e
BHATTACHARJEE, 2012; MYSORE, 2013).
23
Os compostos de sinalização como ROS podem agir diretamente como
toxinas para o patógeno ou desencadear uma resposta sistêmica adquirida
(SCHEEL, 1998). A RSA é não específica e induz alterações bioquímicas e
fisiológicas em plantas, tais como fortalecimento físico da parede celular
através de lignificação, suberificação, e deposição de calose; e produzindo
compostos fenólicos (BOWLES, 1990). Outras respostas envolvem a produção
e acúmulo de fitoalexinas, que são principalmente produzidas por células
saudáveis localizadas adjacentes às células danificadas e necróticas; e
proteínas relacionadas à patogênese (PR) que se acumulam localmente nos
tecidos infectados, e também em tecidos não infectados em locais distantes do
ponto de infecção inicial (RYALS et al., 1996; DURRANT e DONG, 2004).
Figura 3. Resumo esquemático dos processos metabólicos envolvidos no mecanismo de
defesa das plantas ao ataque de patógenos (Adaptado de TORRES, JONES e DANGL,
2006).
24
3.1. Patossistema T. cacao x M. perniciosa
Diversos estudos envolvendo o patossistema M. perniciosa X T. cacao
buscam esclarecer processos bioquímicos e metabólicos que ocorrem durante
a infecção da planta, visando controlar a doença e, consequentemente, reduzir
os prejuízos constantes que a cultura do cacaueiro vem sofrendo ao longo dos
anos.
Ceita et al. (2007) realizaram amplo estudo e observaram diferenças nos
níveis de cristais de oxalato de cálcio entre genótipos susceptíveis e resistentes
a vassoura-de-bruxa que poderiam estar envolvidos com o desenvolvimento da
doença. Dias et al., (2011) analisaram duas variedades de cacau, susceptível e
resistente a H2O2, e o conteúdo de Ácido Oxálico livre e Ácido ascórbico como
o principal precursor de Ácido Oxálico e demonstraram que a quantidade de
cristais de Oxalato de Cálcio e os níveis de H2O2 apresentaram padrões
temporais e genótipo-dependente distintos. Scarpari et al (2005) observaram
alterações bioquímicas associadas com a infecção sugerindo a ação de
mecanismos inespecíficos para tentar eliminar o fungo, como o aumento de
alcalóides, compostos fenólicos e taninos, que são utilizados pela planta.
Gesteira et al., (2007) construíram duas bibliotecas de cDNA, sendo
cada uma delas a partir de cultivares de cacau resistente e susceptível a M.
perniciosa, respectivamente, na busca por um melhor entendimento dos
processos envolvidos nesta interação planta-patógeno, o que possibilitou a
identificação de genes do patógeno e do hospedeiro. Desta forma foi isolado o
gene relacionado à patogenicidade TcPR-10 (Pathogenesis-related protein 10
de Theobroma cacao) (GESTEIRA et al., 2007). Pungartnik et al, (2009a)
relataram o primeiro caso da atividade de ribonuclease e antifúngica da
proteína heteróloga TcPR-10 (TcPR-10p) contra M. perniciosa e
Saccharomyces cerevisiae. Este trabalho demonstrou a forte ação de
ribonuclease contra RNA de M. perniciosa, sendo esta atividade caracterizada
como uma resposta dose e tempo dependente, e os ensaios in vitro de
atividade antifúngica mostraram que a proteína heteróloga TcPR-10 inibe o
crescimento de M. perniciosa. Mutantes de S. cerevisiae para genes de
reparação do DNA, transporte de membrana, transporte de metais e defesas
25
antioxidantes foram expostos a TcPR-10p. Os resultados sugerem um
transporte ativo desta proteína em células de levedura em fase logaritmica de
reprodução e que a atividade antifúngica pode estar associada à
transportadores de membrana e à ação de permeases.
4. Proteínas relacionadas à patogênese (Proteínas PR)
As proteínas PR foram relatadas pela primeira vez em plantas de tabaco
infectadas com vírus do mosaico do tabaco (VAN LOON e VAN KAMMEN,
1970). Posteriormente, estas proteínas foram encontradas em várias plantas.
Proteínas PR, dependendo dos seus pontos isoelétricos, podem ser ácidas
ou básicas, porém com funções semelhantes. Proteínas PR ácidas estão
localizadas nos espaços intercelulares, enquanto que as básicas estão
predominantemente localizadas no vacúolo. Além disso, caracterizam-se pelo
baixo peso molecular (150-163 kDa) e resistência à protease (LEGRAND et al.,
1987; NIKI et al., 1998; VAN LOON e VAN STRIEN, 1999).
Atualmente as proteínas PR são classificadas em 17 famílias de acordo
com a massa molecular, ponto isoelétrico, localização e atividade biológica
(Tabela 1)(LIU e EKRAMODDOULLAH, 2006; VAN LOON, REP e PIETERSE,
2006). Estas famílias incluem inibidores de proteinase (PR6), quitinases (PR3,
4), peroxidases (PR9), proteínas de transferência de lipídios (PR14),
endoprotinases (PR7) e defensinas (PR12) (LIU e EKRAMODDOULLAH,
2006).
26
Tabela 1. Famílias de proteínas relacionadas à patogênese (PR).
Famílias Membros Propriedade Referência
PR-1 PR-1a Tabaco Antifúngico MITSUHARA et al. (2008)
PR-2 PR-2 Tabaco β- 1,3 – glucanase BALASUBRAMANIAN et al.
(2012)
PR-3 P, Q Tabaco Quitinase tipo I, II, IV, V, VI, VII EBRAHIM e SINGH (2011)
PR-4 R Tabaco Quitinase tipo I, II, atividade
ribonuclease
e antifúngica
LU et al. (2012)
PR-5 S Tabaco Thaumatin-like 9 (Antifúngico)
Osmotina
Atividade antifúngica
LOUIS e ROY (2010)
PR-6 Inibidor I de Tomate Inibidor de proteinase LALUK e MENGISTE
(2011)
PR-7 P69 Tomate Endoproteinase TIAN et al. (2004)
PR-8 Quitinase de pepino Quitinase tipo III e atividade
lisozima
SELITRENNIKOFF (2001)
PR-9 “Lignin forming
peroxidase” Tabaco
Peroxidase VAN LOON; VAN STRIEN
(1999)
PR-10 “PR1” de salsa Atividade Ribonuclease,
antifúngica e Dnase
FERNANDES et al. (2013)
PR-11 Quitinase de tabaco
tipo V
Quitinase tipo I VAN LOON; REP;
PIETERSE (2006)
PR-12 Rs-AFP3 Rabanete Defensina AHMED, PARK E JUNG et
al. (2012)
PR-13 Quitinase de tabaco
tipo V
Tionina CHANDRASHEKHARA et
al. (2010)
PR-14 LTp4 cevada LTP (Lipid-transfer protein) EGGER et al. (2010)
PR-15 OxOa cevada
(germin)
Oxalato oxidase SUDISHA et al. (2012)
PR-16 OxOLP cevada Oxalato oxidase-like SUDISHA et al. (2012)
PR-17 PRp27 de tabaco Peptidase CHRISTENSEN et al.
(2002)
27
4.1. Proteínas relacionadas a patogênese da família 10 –PR10
Dentre as proteínas PR destacamos a família 10 cujo mecanismo de
ação, apesar dos diversos estudos realizados, ainda necessita ser
aprofundado. Sabe-se que as proteínas PR-10 são expressas em mono e
dicotiledôneas quando infectados por fungos, oomicetos, vírus, bactérias,
nematóides ou pelo ataque de insetos. São também induzidas em resposta a
indutores de defesa como ácido salicílico (SA), ácido jasmônico (JA) e etileno
(ET). Além disso alguns representantes dessa família são constitutivamente
expressos indicando um papel no desenvolvimento geral da planta (VAN
LOON, REP e PIETERSE, 2006; DOORNBOS et al., 2011).
Proteínas PR-10 são codificadas por famílias multigênicas o que explica
seu caráter multifuncional. Esta característica tem sido recentemente atribuída
a um processo chamado promiscuidade protéica em que os genes adquirem
mutações e funções diferentes ao longo do processo evolutivo (TOKURIKI e
TAWFIK, 2009; FRANCO, 2011). Atualmente já foram descritos mais de 100
representantes dentro da família PR-10 em 70 espécies mono e dicotiledôneas
(WEN et al., 1997; COLDITZ, NIEHAUS e KRAJINSKI, 2007).
Genes PR-10 geralmente consistem de dois éxons interrompidos por um
íntron conservado de 76-359 bps e ORF variando entre 465-480 bps
(HANDSCHUH et al., 2007; LEBEL et al., 2010b). Todos os membros desta
família possuem um motivo rico em glicina altamente conservado denominado
de “p loop motif” (GXGGXGXXK; 47–55 aminoácidos) que está diretamente
relacionado com atividade ribonuclease (CHADHA e DAS, 2006; LYTLE et al.,
2009). Essas proteínas PR-10 apresentam sítios de fosforilação que são
característicos de quinases dependente de cAMP (BANTIGNIES et al., 2000).
A atividade RNAse de PR-10 foi primeiramente observada em cultura de
células de calos de Panax ginseng com uma identidade de 60-70% com duas
proteínas relacionadas a patogênese intracelulares (IPR- Intracellular
pathogenesis related) (MOISEYEV et al., 1994). A presença de 3 sítios comuns
de fosforilação nas proteínas PR10s indicam atividade de RNase geral ou
talvez como RNase específica contra RNA exógeno (GRAHAM et al., 2003;
PARK et al., 2004; KIM et al., 2008).
28
Trabalhos recentes identificaram atividade DNAse das proteínas PR10
em arroz (KIM et al., 2011) e uva (HE et al., 2012) sugerindo uma ação de
nuclease no processo de morte celular programada.
Além das descritas acima, as proteínas PR-10 proteínas possuem outras
funções conhecidas, que não se aplicam a todos os membros do grupo, como
atuação na biossíntese de metabolitos secundários, atividade antimicrobiana,
ligação a membranas como fitohormônios e ligantes hidrofóbicos, estoque,
transporte, dentre outros (FERNANDES et al.,2013).
5. Ferramentas proteômicas na interação planta X patógeno
A análise proteômica envolve a avaliação em larga escala de proteínas
incluindo suas interações, localizações, funções e possíveis modificações. O
desenvolvimento das técnicas de espectrometria de massas possibilitou a
identificação e caracterização de proteínas e seu constante aperfeiçoamento
permite uma maior qualidade dos resultados obtidos principalmente quanto a
resolução, sensibilidade e rendimento (COIRAS et al., 2008).
A utilização da técnica de gel bidimensional (2DE), com inúmeras
proteínas diferencialmente expressas sendo identificadas, tem sido feita com
sucesso em estudos envolvendo a resposta de fungos a drogas. Apesar do
desenvolvimento de técnicas de maior rendimento, diversos estudos baseados
em géis bidimensionais continuam bastante responsivos.
Diversos estudos têm sido realizados buscando identificar proteínas
diferencialmente expressas em micro-organismos relacionadas a etapas do
desenvolvimento, resposta a drogas, proteínas de parede celular, dentre
outras. Groot et al. (2004) analisaram proteínas ligadas a parede celular de
Candida albicans e, utilizando espectrometria de massas, identificaram 14 que
foram divididas em cinco catergorias funcionais: 5 enzimas relacionadas a
carboidratos, 2 proteinas de adesão, 2 semelhantes a superóxido dismutase
que parecem estar envolvida na neutralização de reposta de defesa do
hospedeiro; e as 5 restantes com função desconhecida. Singh et al. (2012)
avaliando a resposta de defesa de Aspergillus fumigatus quando tratado com
29
coumarina, um potente antifúngico, identificaram 143 proteínas
diferencialmente expressas, sendo 13 super-expressas e 96 reprimidas. As
proteínas encontradas estavam envolvidas no controle da divisão celular,
ubiquitinação, ATP sintase vacuolar do tipo A, dentre outras.
Mares (2012) avaliou as proteínas expressas em M. perniciosa durante a
germinação dos esporos nos períodos de 0, 2 e 4 horas através de 2D-PAGE
combinada à Espectrometria de Massa (ms/ms), e observou 514, 434 e 508
spots, respectivamente, nos períodos aval iados. Foram identif icadas
168 proteínas das quais, no período de 4h, foram relacionadas ao metabolismo
energético essencial ao processo de diferenciação hifal. Nos períodos de 2 e 4
horas foram expressas principalmente proteínas associadas a síntese proteica
(MARES, 2012). Lopes et al., (2008) caracterizaram rotas metabólicas
exclusivas do fungo no momento da interação como a rota da quitina. Alvim et
al. (2009) analisaram proteínas secretadas relacionadas com a morfologia da
hifa.
A identificação de proteínas do fungo expressas em resposta a drogas,
como TcPR-10, podem fornecer informações importantes sobre o papel que
desempenham no processo de resposta de defesa do fungo.
6. Saccharomyces cerevisiae: Transporte ABC, formação de vacúolos
e autofagia
Saccharomyces cerevisiae foi o primeiro organismo eucariótico
completamente sequenciado, anotado, e disponibilizado ao público.
(GOFFEAU et al., 1997). Além de sua importância industrial, S. cerevisiae
serve como um organismo modelo para a compreensão da função de células
eucarióticas. Seus genes, distribuídos em 16 cromossomos, apresentam
grande homologia com genes eucariotos sendo, inclusive, capazes de
complementar sua função (BOTSTEIN, CHERVITZ e CHERRY, 1997;
FRIEDBERG, 2006). Dentre os genes de S. cerevisiae destacamos aqueles de
30
interesse nesse esudo: transporte do tipo ABC, autofagia e formação de
vacúolos.
Transportadores de membrana do tipo ABC
O transportadores do tipo ABC são uma superfamília de proteínas que
inclui membros importadores e exportadores. Estas proteínas convertem a
energia obtida pela hidrólise de ATP em um movimento trans-bicamada
levando ao transporte de substratos para dentro do citoplasma (Importação) ou
para fora (Exportação). Os importadores foram encontrados apenas em
procariotos, até o momento, enquanto as proteínas exportadoras são
expressas em todos os reinos (GOTTESMAN e AMBUDKAR, 2001).
Transportadores ABC estão envolvidos em vários processos celulares
como manutenção da homeostase osmótica, processos anti-envelhecimento,
divisão celular, resistência a drogas, patogênese e esporulação, tráfico de
colesterol e lipídeos dentre outros (GEORGE e JONES, 2012). Além destes
existem os transportadores envolvidos com o efluxo de substâncias nocivas a
célula realizando um processo de detoxificação celular através de diferentes
proteínas cuja superexpressão indica a resistência a drogas. Pohl et al. (2012)
analisaram uma população de carrapatos Rhipicephalus (Boophilus) microplus
(Jaguar) resistente a quatro classes de acaricidas. Foi identificado um
mecanismo de desintoxicação baseado em transportadores ABC sugerindo que
estas proteínas atuam na proteção contra vários tipos de acaricidas e
apresentam um importante papel para o desenvolvimento de futuros fármacos.
A levedura S. cerevisiae contém mais de 30 genes do tipo ABC
(DECOTTIGNIES e GOFFEAU, 1997; BAUER; WOLFGER; KUCHLER, 1999)
dentre os quais destacam-se os genes da subfamília PDR (Resistência
Pleiotrópica a Drogas) que codificam para uma complexa rede de reguladores
de transcrição que controlam a expressão de algumas bombas de efluxo de
drogas (WOLFGER; MAMNUN; KUCHLER, 2001). A deleção de
transportadores de membrana nas linhagens mutantes de S. cerevisiae é uma
importante ferramenta para identificação de vias de eliminação de substancias
como, por exemplo, compostos antifúngicos e antibióticos.
31
Tabela 2. Descrição de bombas de efluxo de drogas da família ABC encontradas em
Saccharomyces cerevisiae (Adaptado de PAUMI et al., 2009)
Transportador ORF Localização Descrição
PDR5 YOR153w Membrana plasmática
Transporte múltiplo de drogas
envolvidas na resistência a
compostos xenobióticos;
transporte de cálcio e esteróis
PDR15 YDR406w Membrana plasmática
Transporte de múltiplas drogas
envolvido na resposta geral ao
estresse para detoxificação
celular
PDR10 YOR328w Membrana plasmática
Transporte de múltiplas drogas
envolvido na rede de resistência
de drogas pleiotrópicas
SNQ2 YDR011w Membrana plasmática Transporte de múltiplas drogas
envolvido na resistência a ROS
PDR18 YNR070w Membrana plasmática
Provável transporte que implica
na resistência de drogas
pleiotrópicas
PDR12 YPL058c Membrana plasmática
Transporte de múltiplas drogas
envolvido na resistência a ácidos
organicos.
PDR11 YIL013c Membrana plasmática
Transporte de múltiplas drogas
envolvido na rede de resistência
a drogas e absorção de esteróis
AUS1 YOR011w Membrana plasmática Envolvido com a absorção de
esteróis
YOL075c YOL075c Membrana plasmática Desconhecido
ADP1 YCR011c Membrana plasmática Desconhecido
32
Autofagia
A autofagia (ATG) refere-se a um sistema intracelular de degradação
celular que envolve a entrega de compostos celulares para o
lisossomo/vacúolo. As subunidades geradas após degradação são então
reaproveitadas pela célula, estabelecendo assim uma via de retroalimentação
que permite a manutenção da homeostase (YORIMITSU et al., 2007; WANG;
KLIONSKY, 2011).
Foram identificados três tipos de autofagia: Mediada por chaperonas,
microautofagia e macroautofagia, que diferem entre si quanto a suas funções
fisiológicas e o modo de entrega. Na autofagia mediada por chaperonas,
proteínas malformadas são marcadas por chaperonas, como hsp70, e enviadas
diretamente para o lisossomo; A microautofagia consiste na degradação de
componentes celulares que são diretamente encaminhados ao lisossomo e
então internalizados por invaginação ou protrusão da membrana do lisossomo.
Finalmente, a macroautofagia é caracterizada pelo isolamento do material a ser
degradado em uma estrutura dupla membrana denominada autofagossomo,
também chamado vacúolo autofágico, que se fusiona ao lisossomo, formando o
autolisossomo (Figura 4) (BAEHRECKE, 2005).
O processo autofágico engloba a atuação de 36 proteínas descritas até
o momento, que interagem nas diferentes etapas da rota autofágica. Estudos
de fracionamento celular demonstraram que ATG8 é a principal proteína do
processo de autofagia por estar envolvida no transporte de lipídeos até a
membrana em expansão e estar ligada ao processo de fechamento da
membrana. Mutantes para o gene .ATG8 são incapazes de gerar autofagia
(KIRISAKO et al., 1999). Desta forma a utilização da proteína ATG8 é uma
alternativa para o estudo da dinâmica de membrana durante autofagia.
Pereira (2012) utilizou S. cerevisiae para caracterização da proteína Atg8 para
observar a resistência em diferentes concentrações de tunicamicina (0, 0,2, 0,4
e 0,8 ug / mL) e ditiotreitol (0, 2, 4 e 6 mM / mL) em duas fontes de carbono
(glucose e glicerol), e determinar o perfil de crescimento dos isolados quando
cultivados em glucose e glicerol, a produção da espécies reativas a oxigênio
(ROS) e formação do autofagossomo. Foram testadas quatro linhagens
isogênicas de S. cerevisiae: (A) selvagem (Atg8), (B) selvagem contendo uma
33
cópia do gene ATG8 de M. perniciosa, (C) mutante atg8Δ (gene Atg8 de S.
cerevisiae ausente) contendo uma cópia do gene de ATG8 de M. perniciosa e
(D) atg8Δ mutante. Os resultados obtidos apresentaram diferença fenotípica
entre DXA, DxB e CXD, bem como similaridade de C entre A e B, indicando
uma expressão heteróloga possível da proteína MpAtg8 (Proteína Atg8 de M.
perniciosa).
Figura 4. Representação esquemática das etapas relacionadas as rotas
autofágicas. Macroautofagia (01) Início da formação da membrana de
isolamento (IM). (02) IM totalmente formada originando o autofagossomo que
contém os compostos a serem degradados, (03) formação do autofagolisossomo
através da fusão entre o autofagossomo e o lisossomo, (04) digestão dos
componentes citoplasmáticos via hidrolases lisossomais, (5) Autofagia mediado
por chaperones, (6) microautofagia. (Adaptado de He e Klionsky, 2009).
34
Os genes envolvidos no processo de autofagia estão presentes em
micro-organismos, plantas e animais, e alguns genes autofágicos (ATGs)
conhecidos são bastante conservados nestes grupos. Estes genes atuam na
regulação das vias autofágicas dentre os quais destaca-se o ATG8 (KIEL,
2010).
Poucos estudos têm sido realizados visando observar autofagia em
fungos filamentosos, como M. perniciosa. Pungartnik et al., (2009)
demonstraram que ocorre uma indução transitória do gene MpATG8 em
resposta a estresse oxidativo, assim como foi observado uma variação da
indução de acordo a fonte de carbono utilizada para crescimento, sendo que,
durante as diferentes fases do ciclo de M. perniciosa houve uma expressão
continua da proteína Atg8.
Em S. cerevisiae a proteína Atg8 apresenta 117 aminoácidos que atuam
no início da formação e expansão da IM. Para que ocorra a expansão da PAS
e consequentemente formação da IM e do autofagossomo, lipídeos devem ser
incorporados a estrutura em formação. Para essa adição ser realizada, faz-se
necessário o transporte dos lipídeos até o local de fixação, e este transporte é
efetuado via Atg8p (KIRISAKO et al., 1999; 2000; XIE et al., 2008). Sendo que
o tamanho do autofagossomo formado será proporcional a quantidade de
Atg8p (XIE et al., 2008). Além de estar envolvido no transporte de lipídeos até a
membrana em expansão, estudos de fracionamento celular também
demonstraram que Atg8p é a principal proteína do processo de fechamento da
membrana (KIRISAKO et al., 2000). Em conjunto, estas características tornam
Atg8p uma ferramenta chave para analisar a dinâmica da membrana durante o
processo autofágico.
Sistema vacuolar
O vacúolo é um compartimento acídico importante para células fúngicas
por estar diretamente envolvida com a fisiologia desses organismos. Entre as
muitas funções desta organela estão a manutenção do pH e osmorregulação, a
degradação de proteínas, esporulação, reciclagem de proteínas
armazenamento de aminoácidos, transporte de íons, dentre outros. O vacúolo
35
atua na hidrólise como no lisossomo de mamíferos, e na homeostase,
armazenamento e osmoregulação semelhante ao que ocorre nas plantas
(KLIONSKY, HERMAN e EMR, 1990; TETER e KLIONSKY, 2000).
A biogênese do vacúolo envolve diferentes vias incluindo (i) a triagem
das proteínas vacuolares à distância do local de entrega (ii) a endocitose de
material a partir da membrana plasmática, (iii) segmentação do citoplasma
evitando estágios iniciais da via secretora, e (iv) a herança de material de
vacuolar por células filhas durante a divisão celular (Figura 5).
Defeitos nos vacúolos causados pela exposição a drogas levam a
acidificação e incapacidade desta organela em executar suas funções como
por exemplo incapacitando o organismo de responder ao stress osmótico e
incapacidade de degradar e reciclar componentes celulares danificados
podendo, desta forma, causar sensibilidade aumentada a drogas
(MARKOVICH et al., 2004).
Figura 5. Transporte de proteínas para o vacúolo em leveduras. 1. (a)
Complexo de Golgi através de um compartimento pré-vacuolar (PVC) e
(b) através de um percurso alternativo; 2. endocitose de proteínas da
superfície celular; 3. via biossintética vacuolar; 4. autofagia; 5. Fusão
celular (BRYAN e STEVENS, 1998)
36
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42
IV. Capítulo 1
Identification of differentially expressed proteins in the pathogen Moniliophthora
perniciosa after contact with the antifungal protein TcPR-10
SILVA, F.A.C.1; PIROVANI, C.P.
1, MENEZES, S.
1; PUNGARTNIK, C.
2; SANTIAGO,
A.S. 3; MICHELI, F.
4; GESTEIRA, A.S
5.
1 Laboratório de Biologia Molecular,
2 Laboratório de Biologia de Fungos, Centro de
Biotecnologia e Genética. Universidade Estadual de Santa Cruz. Rod. Ilhéus Itabuna, km
16, Salobrinho, Ilhéus, Bahia, Brasil CEP 45662-900
3 Departamento de Bioquímica, Universidade Federal de São Paulo- Escola Paulista de
Medicina. Rua Sena Madureira, 1500, 5º andar, CEP 04021-001, São Paulo, Brasil
4 CIRAD, UMR AGAP, F-34398 Montpellier, France
5 Embrapa Mandioca e Fruticultura, Departamento de Biologia Molecular, Rua Embrapa,
s/nº, CEP44380-000; Cruz das Almas, Bahia, Brasil
Submtido ao periódico Molecular Biology Reports (Fator de Impacto: 2.92)
Corresponding author: Abelmon da Silva Gesteira. Embrapa Mandioca e
Fruticultura, Rua Embrapa, s/nº, 44380-000, telefone/fax; Cruz das Almas, Bahia,
Brasil. E-mail: [email protected].
43
ABSTRACT
The TcPR-10 protein, which is related to the pathogenesis of family 10, was discovered
from a library of interactions between Theobroma cacao and Moniliophthora perniciosa.
TcPR-10 has been shown to have antifungal and ribonuclease activities in vitro. This study
aimed to identify differentially expressed proteins in Moniliophthora perniciosa in
response to the antifungal activity of TcPR-10 through proteomic analysis. The fungal
hyphae were subjected to four treatments: a control treatment (without TcPR-10) and 30
min, 60 min or 120 min treatments with TcPR-10 protein. Two-dimensional maps
identified 191 differentially expressed proteins, of which 55 were identified by mass
spectrometry. The identified proteins in all four treatments were divided into the following
classes: cell metabolism, stress response, zinc binding, phosphorylation mechanism,
transport, autophagy, DNA repair, and oxidoreductases. The predominant class was stress
response proteins (29%), such as 14-3-3 and heat shock proteins (HSP), which had the
highest expression levels in the control treatment and are known to trigger defense
mechanisms against drugs, such as TcPR-10. Oxidoreductases (25%) were overexpressed
in the control and 30 min treatments but exhibited reduced expression at 120 min. These
proteins are involved in the repair of damage caused by oxidative stress from contact with
TcPR-10. Given the antifungal activity of TcPR-10, several identified proteins were related
to detoxification or autophagy or were involved in mechanisms for maintaining fungal
homeostasis, such as in ergosterol biosynthesis. The results show that the sensitivity of the
fungus M. perniciosa to TcPR-10 involves several biochemical routes that clarify possible
modes of action for these antifungal proteins.
Keywords
Pathogenesis-10-related protein, differentially expressed proteins; stress response proteins;
oxidoreductases
44
INTRODUCTION
Moniliophthora perniciosa (Basiomicota, Agaricales, Marasmiaceae) is the causal
agent of the disease in the cacao tree (Theobroma cacao L.) called “witches' broom”
(AIME; PHILLIPS-MORA, 2005). This disease is considered the most important cause of
economic loss in South America and the Caribbean, causing great damage to cocoa
plantations and affecting the production of this commodity (KILARU; HASENSTEIN,
2005; SCARPARI et al., 2005).
The life cycle of M. perniciosa is hemibiotrophic and is divided into two phases:
the biotrophic (parasitic) phase, which is characterized by monokaryotic hyphae,
hyperplasia and hypertrophy of plant tissue, loss of apical dominance, axillary shoot
proliferation, and abnormal stems (green broom); and the necrotrophic (saprophytic) phase,
which is characterized by dikaryotic hyphae containing clamp connections and necrosis
and death of infected tissues distant from the primary infection (broom dry). Basidiocarp
production and spore formation in the infected necrotic tissue mycelium occur on the
surface of plant tissue or, more often, inside the plant in direct contact with the plasma of
the host (AIME; PHILLIPS-MORA, 2005; SCARPARI et al., 2005; MEINHARDT, L.W.
et al., 2008).
The great damage caused by witches' broom has motivated several genomic and
proteomic studies aiming to better understand the physiology of this pathogen and its
mechanism of attack on cocoa. Gesteira (GESTEIRA et al., 2007) constructed two cDNA
libraries, each from cocoa cultivars that are resistant and susceptible to M. perniciosa, and
identified the gene related to pathogenicity, TcPR10 (Pathogenesis-related protein 10 of
Theobroma cacao). TcPR-10 is a member of a family of acidic proteins, PR-10, that are
found in some gymnosperms and angiosperms and that are responsive to intracellular
defense processes (ISLAM, M. A. et al., 2009; LEBEL et al., 2010a; XIE, Y.R. et al.,
2010). Members of the PR10 family have a highly conserved, rich glycine motif called the
"p loop motif" (GXGGXGXXK; 47-55 amino acids) that is involved in ribonuclease
activity (CHADHA; DAS, 2006; LYTLE et al., 2009). Another feature of the PR10 family
is the presence of phosphorylation sites that are characteristic of cAMP dependent kinases
(BANTIGNIES et al., 2000). The presence of three common phosphorylation sites in PR-
10 proteins suggests a general role with RNase or an RNase specific effect against
exogenous RNA (GRAHAM et al., 2003; PARK et al., 2004; KIM et al., 2008). Recent
45
work identified DNAse activity in PR-10 proteins in rice (KIM, S. G. et al., 2011) and
grapes (HE et al., 2012), suggesting an action of nuclease in programmed cell death.
Pungartnik (PUNGARTNIK; DA SILVA; et al., 2009a)conducted the first report of
the ribonuclease and antifungal activity of the heterologous protein TcPR-10 against M.
perniciosa and Saccharomyces cerevisiae. This work demonstrated a strong, ribonuclease
action of TcPR-10 against the RNA of M. perniciosa, characterized as a concentration- and
time-dependent response, and the in vitro antifungal activity showed that TcPR-10
inhibited the growth of M. perniciosa.
Several studies have investigated the defense mechanisms triggered by plants in
response to pathogen attacks and analyzed the mechanism by which these pathogens
establish themselves in the host tissue. Research has also examined the survival
mechanisms that pathogenic fungi develop in response to either antimicrobial substances
produced by plants or anthropogenic origins, such as pesticides. The use of proteomic tools
to assess the effects of antifungal agents against a fungus has had great applicability for
providing a comprehensive examination of the changes that occur in the pathogen. Gautam
(GAUTAM et al., 2007) and Cagas (CAGAS et al., 2011) investigated the proteomics of
Aspergillus fumigatus in response to stress generated by coumarin, a known antifungal
drug, and the possible mechanism of action by which the compound produced lethal effects
on the fungus. Hoehamer (HOEHAMER et al., 2010) and Bruneau (BRUNEAU et al.,
2003) observed changes in the proteome of Candida albicans after treatment with azole
antifungals, poliene, echinocandin and two triazoles: fluconazole and itraconazole. In this
study, we investigated the mechanism of the antifungal activity TcPR-10 in M. perniciosa
through proteomic analysis.
46
MATERIAL AND METHODS
1. Obtaining the TcPR-10 protein
The cocoa gene TcPR-10 was isolated from the cDNA library interaction T. cacao x
M. perniciosa. The gene was cloned into the expression vector pET28a, and TcPR-10
recombinant protein was expressed in Escherichia coli BL21 (DE) (PUNGARTNIK; DA
SILVA; et al., 2009a; MENEZES, S.P. et al., 2012). The recombinant protein TcPR-10
was concentrated to a stock solution of 30 mg / ml.
2. M. perniciosa growth conditions
The M. perniciosa fungus (strain 553) was grown in 2% agar medium CPD
(Crinipelis Peptone Dextrose). For each replicate of the experiment, five 1 cm diameter
circles were cut out and added to a test tube containing 5 mL of 2% medium CPD and 1 g
of "glass beads" for mechanical breakdown in a hyphal vortex for 1 min. The solution of
broken hyphae was then transferred to an Erlenmeyer flask containing 20 ml of 2% CPD
that was constantly stirred at 100 rpm at 25 ° C for 7 days. After this period, we disturbed
the hyphae formation using a 5 mL pipette. Hyphae supernatants were transferred to new
Erlenmeyer flasks containing 20 ml of 2% CPD medium and kept under constant stirring at
100 rpm at 25 ° C for 7 more days.
3. Exposure of the fungus M. perniciosa to TcPR-10
Recombinant protein TcPR-10 was added to each flask containing hyphae of the
fungus M. perniciosa in 20 ml of 2% CPD medium to reach a final protein concentration of
3 μg/mL. The fungal hyphae were collected either without contact with the protein TcPR-
10 or after 30 min, 1 h and 2 h of exposure to the antifungal protein, rinsed with ultra-pure
water, autoclaved, dried, frozen with liquid N2 and stored in a freezer at -80 ° C.
4. Protein extraction and quantification
Proteins were extracted following the methods in Pirovani (PIROVANI et al.,
2008). Protein extracts were subjected to a second cleaning method based on Meyer
(MEYER et al., 1988), aiming for the purification of total protein. The proteins were then
47
quantified using the 2D Quant Kit (GE Healthcare) according to the manufacturer's
instructions and using BSA as the standard.
5. 2D gel electrophoresis
Isoelectric focusing (IEF) was performed with 350 mg of protein in a final volume
of 250 µl of rehydration solution (7 M urea, 2 M thiourea, 1% CHAPS, 100 mM DTT,
0.5% IPG buffer (pH 3-10) containing a pinch of bromophenol blue. Samples were applied
to 13 cm IPG "strips" with linear pH gradients from 3 to 10 (Amersham Biosciences,
Immobiline Dry-Strip ™) and subjected to isoelectric focusing with a unit III
EthanIPGphor with the following steps: 1:00 h at 500 V, 1:04 h at 1000 V, 2:30 h at 8000
V, and 0:22 h at 8000 V. The current was limited to 50 μA for each strip, and the
temperature was kept at 20 º C for all steps. The strips were treated with 7 ml of
equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8)
containing 1% DTT (Dithiothreitol) for 15 min with slow agitation, and they were then
transferred to equilibration buffer containing 2.5% iodoacetamide under slow stirring for
another 15 min. Finally, the strips were washed with 1X running buffer (25 mM Tris, 192
mM glycine, 0.1% (w / v) SDS, pH 8.3) for 15 min.
The second dimension was performed with 12.5% polyacrylamide gels at 80 V/200
mA using the Hoefer SE 600 vertical Ruby (GE Healthcare) electrophoresis system. The
gels were fixed in solution containing 40% ethanol and 10% acetic acid for 1 h and then
transferred to 0.1% coomassie brilliant blue G-250.
6. Image analysis and spot detection
Gels were prepared in triplicate and compared two by two. The images of the gels
were acquired using an ImageScanner II (GE Healthcare) scanner and analyzed with the
Image Master 2D Platinum v. 7.0 (GE Healthcare) software to assess the relative change in
area and intensity of spots corresponding to proteins expressed in different treatments.
7. Preparation of spots for MS/MS
The differentially expressed spots were excised from the gel, cut into pieces and
placed in microcentrifuge tubes. The gel fragments were then destained in 200 µL of
48
NH4HCO3 containing 50% acetonitrile, the supernatant was discarded, the gel fragments
were dehydrated in 100 mL of acetonitrile 100 % for 5 min and then vacuum dried at 5301
Concentrator (Eppendorf) for 10 min. In all, 4 µL of Gold trypsin (Promega) was added to
the gel fragments at a final concentration of 25 ng / µL and kept at 4 ° C for 10 min to
absorb residual solution in the gel fragments. NH4HCO3 was later added to cover the
pieces, and the mixture was left at 37 ° C for 16 hours for the trypsin reaction to complete.
The supernatant was collected and transferred to a new tube. The gel fragments
were washed twice with 50 µL of 50% acetonitrile containing 0.1% formic acid and stirred
by vortexing for 15 min in each wash. The volumes obtained were then combined and
concentrated under vacuum to reach a volume of 20 µL.
RESULTS AND DISCUSSION
In the present study, we used mass spectroscopy to elucidate the adaptive responses
of M. perniciosa to the antifungal agent TcPR-10. We identified proteins that changed their
abundances after exposure to this agent. Our work expands on our previous study, which
demonstrated that TcPR-10 exhibited ribonucleic and antifungal activities against M.
perniciosa and S. cerevisiae (PUNGARTNIK; DA SILVA; et al., 2009a; MENEZES, S.P.
et al., 2012).
The analysis of 2-DE gels was performed using the Image Master 2D Platinum
software version 7.0. We utilized four treatments: a control treatment (without TcPR-10)
and 30 min, 60 min and 120 min treatments with TcPr10. The two-dimensional maps
displayed a profile of proteins with molecular weights between 3 and 201 kDa and pH
values between 3 and 10. We observed that the control gel contained 69% of spots with pH
values between 3 and 7 and 31% with pH values between 7 and 10; the 30 min treatment
gel contained 61% of spots with pH values between 3 and 7 and 39% with pH values
between 7 and 10; the 60 min treatment gel contained 62% of spots with pH values
between 3 and 7 and 38% with pH values between 7 and 10; and the 120 min treatment gel
contained 50% of spots with pH values between 3 and 7 and 50% with pH values between
7 and 10 (Figure 1). The differentially expressed proteins were identified from the
following pairwise combinations of these treatments: control x 30 min; control x 60 min;
control x 120 min; 30 min x 60 min; 30 min x 60 min and 60 min x 120 min. The number
49
of protein spots from each treatment, the number of differentially expressed proteins in
each combination and exclusive proteins are listed in Table 1.
The Image Master software identified a total of 191 differentially expressed
proteins between all treatments. There were changes in the expression patterns of proteins,
both under- and overexpressed, and changes in the relative molecular weights and
isoelectric points of proteins. The mass spectrometry analysis identified 55 differentially
expressed proteins. All proteins were differentially expressed in at least three pairwise
comparisons and had an oscillatory expression pattern after 0 h (Control), 30 min, 60 min
and 120 min of exposure to TcPR-10 (Table 2; Figures 1).
In the initial periods of 0 h and 30 min of exposure to TcPR-10, there was an
overexpression of oxidoreductases (Proteins 18, 82, 108, 109, 111, 112, 120, 121, 132 and
148). Oxidoreductases are involved in antioxidant defense, maintenance of intracellular
redox balance and repair of damage caused by oxidative stress. These enzymes have
different mechanisms of action, e.g., oxidases function by electron transfer, hydrogenases
and dehydrogenases function by transfer of hydrogen atoms, oxygenases function by
transfer of oxygen atoms and hydroperoxidases function by degradation of hydrogen
peroxide. Mur (MUR et al., 2004) observed that PR-10 gene expression was influenced by
the presence of reactive oxygen species (ROS) and observed overexpression of AoPR-10-
GUS in transgenic Arabidopsis thaliana when the plants were exposed to salicylic acid, an
important inducer of ROS, or in the presence of virulent bacteria. We note that
oxidoreductases were expressed by fungi to withstand the oxidative damage caused by
TcPR-10, reducing the expression of TcPR-10 at 120 min. These results indicate that a
ROS have a potential negative influence on TcPR-10 expression.
Reactive oxygen species, which are highly potent oxidants, are produced as
products during normal metabolic processes or by an unfavorable environment. Within the
cell, ROS react with macromolecules, including lipids, carbohydrates, DNA and proteins,
to trigger molecular damage, such as DNA mutations, lipid peroxidation, and protein
oxidations, eventually leading to cell death and progressive aging of the organism
(BLACKMAN et al., 2011; HELLER; TUDZYNSKI, 2011). Eukaryotic cells contain
mechanisms to respond to and protect from stress conditions, including neutralization of
the stress, pausing of the cell cycle, alterations in translation, and repair of damage to
apoptotic or necrotic pathways (THOMPSON et al., 2008). The stress caused in cell fungi
50
by drugs, such as TcPR-10, triggers some defense mechanisms against oxidative stress, of
which antioxidants are relevant endpoints.
Malate dehydrogenase (spot 82, 109) is an oxidoreductase that catalyzes the
interconversion of oxaloacetate to malate and participates in the metabolic processing of
carbohydrates, in cellular aging and in the tricarboxylic acid cycle (NICHOLLS and
GOWARD, 1994). Glyceraldehyde-3-phosphate dehydrogenase (spot 18, 102, 108, 111,
112, 121) is a key glycolytic enzyme that exists primarily in the cytoplasm (HARA et al.,
2005), but when in the nucleus, it plays a role in gene transcription, DNA replication, DNA
repair, and nuclear RNA export (ZHENG; ROEDER; LUO, 2003; HARA et al., 2005).
The enzyme 6-phosphogluconate dehydrogenase, decarboxylating 1 (6PGDH) (spot 132)
catalyzes the rate-limiting, NADPH-producing step in the pentose phosphate pathway.
6PGDH has an important role in the acquisition of tolerance against oxidative stress
(IZAWA et al., 1998). Catalase (spot 148) is an antioxidant system located in peroxisomes,
cytosol and spores, that has been implicated in overcoming the host defense response in
several phytopathogens (PERAZA; HANSBERG, 2002; BLACKMAN et al., 2011).
We identified two proteins related to the process of autophagy 30 min and 60 min
after treatment with TcPR-10, GTP-binding nuclear protein and autophagy-related protein.
Autophagy is the process by which intracellular lysosomal degradation compounds are sent
for cellular degradation in the lysosome and vacuoles, and subunits generated after
degradation are then reused by the cell, thereby establishing a feedback path that allows for
the maintenance of homeostasis (YORIMITSU et al., 2007). Reactive oxygen species are
stress-inducing signaling molecules during autophagy (SCHERZ-SHOUVAL et al., 2007),
and therefore, the action of the protein TcPR-10 on M. perniciosa may be related to the
expression of the GTP-binding nuclear protein and autophagy-related protein in all of the
analyzed time periods in an attempt to detoxify the cell. According Pozuelo-Rubio
(POZUELO-RUBIO, 2012), autophagy is modulated by 14-3-3 proteins that showed
highest expression in the control treatment (0 h) and a decreased expression over time.
Conversely, markers for autophagy had their highest expression at 60 min.
Two proteins involved in the repair of DNA sequence, Ubiquitin-conjugating
enzyme and the Nucleoside diphosphate kinase Ndk1, were also identified. The Ubiquitin-
conjugating enzyme exhibited higher expression at 60 and 120 min, whereas Ndk1 showed
greater activity at 30 min. Kim (KIM, S. G. et al., 2011) and He (HE et al., 2012)
demonstrated that PR-10 has DNAase activity that triggers the expression of protein repair
51
enzymes. Ndk1 repairs the DNA sequence by replenishing lost nucleotides (CHOPRA et
al., 2003), whereas proteins regulating ubiquitination in the repair of double-stranded DNA
are caused by toxic substances (WU et al., 2003; MOUDRY et al., 2012), such as TcPR-
10.
Most proteins identified were classified as stress responsive (spots 8, 20, 24, 28, 33,
45, 46, 48, 85, 87, 102, 105, 153, 162, 170 and 184). Among these proteins, 14-3-3
proteins showed the highest expression level in the control treatment (0 h), as 14-3-3
proteins have conserved regulatory activities in eukaryotic cells, such as kinases,
phosphatases and transmembrane receptors. Moreover, 14-3-3 proteins are notable for
playing important roles in cell death through apoptosis, cell cycle control and stress
response (FU; SUBRAMANIAN; MASTERS, 2000). Heat shock proteins (HSP) (spot 24,
45, 48, 85, 87, 153 and 170) are molecular chaperones that are found in all organisms and
are natural defensive mechanisms in response to various types of stress, e.g., oxidative
stress caused by drugs or environmental changes (BURNIE et al., 2006; WANDINGER;
RICHTER; BUCHNER, 2008).
Among the proteins expressed by the fungus in response to TcPR-10-induced stress
were proteins involved in maintenance of fungal wall integrity against oxidative damage
caused by the antifungal protein. The alpha-1 subunit of the 26S proteasome is an ATP-
dependent protease that prevents the accumulation of degraded proteins (YANG et al.,
2004; VIERSTRA, 2009) and therefore provides greater resistance to oxidative stress.
However, it is possible that the observed decrease in its expression over time correlated
with an increase in the amount of oxidized proteins.
The protein Acetyl-CoA acetyltransferase (spot 22) is involved in ergosterol
biosynthesis in fungi. Ergosterol regulates cell membrane fluidity and permeability and is
essential for cell survival (PARKS et al., 1999). Upregulation of ergosterol at 0 and 30 min
denotes a mechanism to preserve fungal membrane integrity and maintain the function of
membrane-bound proteins to avoid an inhibition of fungal growth (RUGE; KORTING;
BORELLI, 2005). Bet v 1, a homolog to the PR-10 protein, has binding sites for fatty
acids, flavonoids, cytokinins, emodin and sterols, suggesting that this protein has a role in
the storage and transport of biologically important molecules with activity in various
tissues and environmental conditions (KUNDU; ROY, 2010).
The proteins in the largest class that responded to TcPR-10 exposure were involved
in several mechanisms (Figure 2). Our functional analysis showed that the majority of
52
differentially expressed proteins identified by mass spectrometry were involved in stress
response (29%) and cell metabolism (22%), indicating a fungal response to maintain
homeostasis and presenting possible mechanisms of action of TcPR-10 upon and after
contact.
Several PR-10 proteins with antifungal activities have been described in the
literature: SsPR-10 (LIU et al., 2005), CsPR-10 (GÓMEZ‐GÓMEZ; RUBIO‐MORAGA;
AHRAZEM, 2010); CaPR-10 (PARK et al., 2004), maize PR-10 proteins (CHEN et al.,
2006; XIE, Y.R. et al., 2010) and JcPR-10a (AGARWAL et al., 2012). Pungartnik
(PUNGARTNIK; DA SILVA; et al., 2009a) used mutants of S. cerevisiae genes for DNA
repair, membrane transport, metal transport and antioxidant defense to demonstrate
ribonucleic and antifungal activities of TcPR-10 against M. perniciosa and Saccharomyces
cerevisiae. This study showed that TcPR-10 actions involving synthesis, modification or
degradation might confer multiple biological functions to individual genes. Proteins were
related with biosynthesis, carbohydrate metabolism, assembly, folding, translational
regulation or amino acid biosynthesis transport. These modifications and their impacts on
virulence, intracellular signal cascades and other biological processes are areas for further
investigation.
In summary, this study describes the first proteomic analysis of the phytopathogenic
fungus M. perniciosa in response to the TcPR-10 protein of cocoa. The identification of a
number of differentially expressed proteins after TcPR-10 contact provides a new
information to unravel the molecular basis of the pathogenesis of these fungi in the
presence of antifungal molecules. The findings in this study of expression of proteins
involved in DNA repair, expression of a protein involved in zinc binding, high expression
of proteins involved in ROS response, and in particular, expression of stress response
proteins together demonstrate the great potential for TcPR10 to be used as an antifungal
agent against M. perniciosa. Further research is required to analyze the behavior of
differentially expressed proteins at other time periods and to identify new proteins involved
in TcPR-10 response.
53
ACKNOWLEDGMENTS
This research supported by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq, Brazil), Fundação de Amparo à Pesquisa da Bahia (FAPESB, Brazil).
F. A.C. Silva was supported by CAPES. Dr. Samuel Saito, Msc. Julian Santana and Leila
Lopes for their help in the identification of proteins by mass spectrometry.
FIGURE LEGENDS
Figure 1. Protein profile in 2-DE gel Moniliophthora perniciosa fungus treated with the
antifungal protein TcPR-10 obtained from cacao. Treatments: A- Control (no TcPR-10); B-
30 min; C- 60 min and D- 120 min after exposure to TcPR-10. The gels were stained with
Coomassie Coloidal Blue G-250 solution. Line MW: molecular weight marker proteins
(kDa). → Excision place gel to differentially expressed proteins identificated by mass
spectrometry.
Figure 2. Representations of the distribution of identified differentially expressed proteins
identified M perniciosa according to their biological process. Categorizations were based
on information provided by the online resource UniProt classification system.
54
Table 1. Distribution of spots between treatments (Control, 30 min, 60 min and 120 min) and between
combinations of these treatments.
Total number of
spots
Differentially expressed
spots Exclusives spots
Control 244 23
30 min 309 58
60 min 298 16
120 min 380 80
Control X 30 min 160
Control X 60 min 118
Control X 120 min 183
30 min X 60 min 173
30 min X 120 min 110
60 min X 120 min 178
55
Table 2. Differentially expressed proteins identified by mass spectrometry (NANO/ESI/Q-TOF).
Match
ID Gel MW PI Access Protein Biologic Process
Control X
30 Control X 60 Control X 120 30 X 60 30 X 120 60 X 120
1 B 45 5.4 P17967 Protein disulfide-isomerase 1 cell redox homeostasis 123.172 0.770709 -0.629766 0.78483 -0.588082 -123.172
3 A 26 7.8 P79071 60S ribosomal protein L6 cytoplasmic translation -0.349022 0.612684 0.600063 -0.612684 -0.193018 -0.191066
8 B 23 8.5 P43773 ATP-dependent protease ATPase
subunit HslU (Heat shock) Stress response -0.74422 114.433 0.849442 -0.881711 -114.433 0.910075
9 A 24 7.8 P40303 Proteasome component PRE6
proteasomal ubiquitin-dependent protein
catabolic process; regulation of mitotic cell
cycle
-0.681787 -0.681787 - - -0.316403 -0.316403
10 B 25 8.1 C6A011 UPF0107 protein TSIB_1943 Phosphorylation -0.38004 -0.441773 0.757425 -0.757425 -0.501307 -0.71635
12 A 16 5.8 P07280 40S ribosomal protein Ribosome biogenesis -0.391769 0.87409 0.704391 -0.770834 -0.906923 0.906923
13 C 15 5.2 O74983 Ubiquitin-conjugating enzyme Postreplication repair -0.793504 0.982094 125.934 -125.934 -0.232689 0.706866
18 B 28 6.8 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase 178.397 0.770195 -0.703513 0.940579 -0.685514 -178.397
19 A 31 6.6 P36010 Nucleoside diphosphate kinase Response to DNA damage stimulus -0.721473 0.803665 -0.832114 0.983847 -0.983847 -0.619165
20 A 32 6.2 P15019 Transaldolase pentose-phosphate shunt (Stress response) -0.539107 0.812164 108.524 -108.524 -0.800265 0.971417
22 A 3
4 6.8 P41338 Acetyl-CoA acetyltransferase Ergosterol biosynthetic process -0.392888 462.225 -0.388934 -0.598483 -462.225 0.983996
24 A 35 4.2 P02829 Heat shock protein 82 Stress response 0.7276 -0.957569 0.828066 -0.585089 0.957569 -0.767368
26 B 35 5.7 P64201 Aspartyl/glutamyl-tRNA(Asn/Gln)
amidotransferase subunit B Protein biosynthesis 0.795523 0.928544 0.908495 -0.886851 -0.9056 -0.928544
28 B 38 6.4 Q9RA63 Chaperone protein ClpB Stress response -0.871045 -0.513589 0.871045 -0.624079 -0.231558 0.591141
32 C 42 5.5 Q10055 ATP-dependent RNA helicase fal1 rRNA processing 0.618478 0.96849 -0.720532 -0.655698 -0.731089 -0.96849
33 C 38 6.4 P32318 Thiazole synthase Suicide enzyme; Stress response and in
DNA damage tolerance. 102.195 0.830277 -0.801889 0.994818 -0.749269 -102.195
34 B 44 5.7 P00830 ATP synthase subunit beta Transport 132.282 0.761055 -0.580592 0.92712 -0.716573 -132.282
36 C 45 6.1 P10507 Mitochondrial-processing peptidase
subunit beta Phosphoprotein 0.951766 0.832967 -0.656623 0.962016 -0.808762 -0.962016
40 A 52 5.6 P16140 V-type proton ATPase subunit B Phosphoprotein -0.584401 0.873055 0.853564 -0.873055 -0.830431 0.839563
44 B 62 4.4 Q52454 Cobalamin synthase zinc ion binding 109.902 -0.546875 0.594864 -0.35976 0.624824 -109.902
45 B 68 5.0 Q01877 Heat shock protein HSS1 Stress response 171.898 0.809091 -0.70584 0.987857 -0.771718 -171.898
56
46 B 75 4.7 P16474 78 kDa glucose-regulated protein
homolog Stress response 112.712 0.671421 0.63484 0.830117 0.792162 -112.712
48 C 84 4.9 P54651 Heat shock cognate 90 kDa protein Stress response -0.577448 0.999935 -0.597114 0.852592 -0.999935 0.663631
50 A 54 9.1 P07251 ATP synthase subunit alpha,
mitochondrial ATP catabolic process -0.803737 243.439 -0.835913 0.749876 -243.439 -0.728872
52 A 58 8.5 Q6FM63 Autophagy-related protein 18 Autophagy -0.924253 18.815 -0.951401 0.973092 -18.815 -0.51398
62 C 21 6.9 Q9AML4 GTP-binding nuclear protein Autophagy -0.419916 - -0.419916 -0.308695 - -0.308695
82 A 31 7.6 P61889 Malate dehydrogenase Oxidoreductase 15.995 0.436343 -0.390386 -0.18428 -0.187161 -15.995
85 A 72 6.7 Q01877 Heat shock protein HSS1 Stress response -0.571641 0.982635 -0.982635 0.568348 -0.691616 -0.71818
87 A 100 6.0 O74225 Heat shock protein Hsp88 Stress response -0.741642 - -0.741642 -0.522303 - -0.522303
92 A 17 5.3 P23301 Eukaryotic translation initiation factor
5A-1 Protein biosynthesis 153.074 0.760242 -0.519375 0.639513 -0.583366 -153.074
94 A 20 5.6 P36010 Nucleoside diphosphate kinase Ndk1 Repair of UV radiation- and etoposide-
induced DNA damage - -0.328555 -0.435905 - -0.435905
102 A 24 5.9 O74770 Probable phosphoketolase Stress response -111.257 - -111.257 -0.877396 - -0.877396
105 B 23 6.9 O42766 14-3-3 protein homolog Stress response 133.195 0.863126 -0.845349 0.935099 -0.580608 -133.195
108 C 30 7.1 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase -0.307501 - -0.307501 -0.567239 - -0.567239
109 C 30 8.0 P17505 Malate dehydrogenase, mitochondrial Oxidoreductase -0.754071 0.801155 -0.89945 0.991879 -0.991879 -0.808336
110 A 32 5,4 P38272 SWI5-dependent HO expression
protein 3 Transport -0.272577 - -0.272577 -0.574921 - -0.574921
111 C 32 7.2 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase 0.849117 0.928823 -100.518 100.518 -0.625291 -0.866885
112 C 32 6.5 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase 0.606736 166.442 -0.615852 0.994095 -166.442 -0.64479
117 A 36 7.0 P49089 Asparagine synthetase [glutamine-
hydrolyzing] 1 Phosphoprotein -0.543259 - -0.543259 -0.79558 - -0.79558
120 B 37 8.3 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase 0.993355 0.915481 -0.795396 0.783463 -0.198078 -0.993355
121 B 38 6.8 P00359 Glyceraldehyde-3-phosphate
dehydrogenase Oxidoreductase -0.742154 0.895575 -0.878184 0.976975 -0.976975 -0.660193
128 B 41 6.5 P39954 Adenosylhomocysteinase Phosphoprotein 0.607278 0.524697 -0.583788 0.909108 -0.766858 -0.909108
129 A 42 7.2 P00560 Phosphoglycerate kinase Phosphoprotein -0.7911 193.703 -0.842342 0.710606 -193.703 -0.715645
130 A 42 6.9 P19358 S-adenosylmethionine synthase 2 One-carbon metabolism 0.827082 136.415 -0.794706 0.905275 -136.415 -0.849288
132 A 45 8.1 P38720 6-phosphogluconate dehydrogenase,
decarboxylating 1 Oxidoreductase -0.348645 - -0.348645 -0.579874 - -0.579874
57
146 A 58 7.7 P39522 Dihydroxy-acid dehydratase,
mitochondrial Amino-acid biosynthesis -0.110582 - -0.110582 -0.464429 - -0.464429
147 B 58 6.7 P40530 Pyruvate kinase Carbohydrate metabolism 0.763106 101.327 -0.61609 0.908803 -101.327 -0.760471
148 C 58 7.9 I2DFD6 Catalase Oxidoreductase -0.382351 - -0.382351 -0.588187 - -0.588187
153 C 64 6.5 P17820 Chaperone protein DnaK Stress response 0.680602 117.191 -0.414151 0.981432 -117.191 -0.730904
162 A 90 7.7 P82610
5-
methyltetrahydropteroyltriglutamate--
homocysteine methyltransferase
Stress response -0.553217 - -0.553217 -0.358378 - -0.358378
170 A 124 4.8 P55737 Heat shock protein 90-2 Stress response -0.311785 0.484568 -0.411449 0.499916 -0.499916 -0.418004
171 A 125 5.1 P25694 Cell division control protein 48 Cell cycle; Autophagy -0.247938 0.549927 -0.285886 -0.369707 -0.549927 -0.525438
179 A 32 5.9 P40825 Alanine--tRNA ligase, mitochondrial Protein biosynthesis - -0.327165 -0.327165 -0.420323 -0.420323 -
184 B 48 6.4 P32318 Thiazole synthase Stress response - -0.789057 -0.789057 -0.381988 -0.381988 -
189 A 97 6.9 Q8MML5 Paxillin-B Zinc ion binding - -0.584424 -0.584424 -0.615015 -0.615015 -
58
FIGURE 1
59
FIGURE 2
22%
11%
5%
29% 4%
20%
4%
5%
Cell metabolism Phosphorilation mecanism DNA repair
Stress response Transport Oxidureductase
Zinc biding Autophagy
60
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62
V. Capítulo 2
Mitigation of oxidative stress induced by TcPR-10 protein in fungi involves
vacuole formation and autophagy
SILVA, F.A.C.1; PUNGARTNIK, C2; PEREIRA, A. C. F2; CARDOSO, T.H.S.3;
MICHELI, F.4; MARQUES, E.C.S1; VERDEIL, J-L. 4; BRENDEL, M2; GESTEIRA,
A.S5.
1 Laboratório de Biologia Molecular, Centro de Biotecnologia e Genética. Universidade Estadual de
Santa Cruz. Rod. Ilhéus Itabuna, km 16, Salobrinho, CEP 45662-900, Ilhéus, Bahia, Brasil.
2 Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética. Universidade Estadual de
Santa Cruz. Rod. Ilhéus Itabuna, km 16, Salobrinho, CEP 45662-900, Ilhéus, Bahia, Brasil.
3 CIRAD, UMR AGAP, F-34398 Montpellier, France .
4 Embrapa Mandioca e Fruticultura, Departamento de Biologia Molecular, Rua Embrapa, s/nº,
CEP44380-000; Cruz das Almas, Bahia, Brazil..
* both authors contributed equally to the manuscript
Running title: PR-10p induces oxi-stress and autophagy in fungi
Submetido ao periódico Plos One (Fator de impacto= 4.03)
Corresponding author: Abelmon da Silva Gesteira. Embrapa Mandioca e
Fruticultura, Rua Embrapa, s/nº, 44380-000, Cruz das Almas, Bahia, Brazil. E-
mail: [email protected]
63
ABSTRACT
The pathogenesis-related protein PR-10 of Theobroma cacao has antifungal action
and ribonuclease activity in vitro. However, the mechanism of transport into the
intracellular medium and its effects within the fungal cell need to be elucidated. We
report the transport of TcPR-10p via the ATP-binding cassette (ABC) transporter
Pdr11p and the involvement of the autophagic process as well as vacuole formation
using Saccharomyces cerevisiae and Moniliophthora perniciosa. Whereas ABC-
transporter mutants yor1Δ, pdr5Δ, pdr10Δ, pdr12Δ and pdr15Δ of S. cerevisiae had
wild-type-like sensitivity to TcPR-10p, isogenic mutant pdr11Δ showed higher-than-
WT resistance. Contrarily, autophagy-deficient mutant atg8Δ showed three-fold
higher sensitivity to TcPR-10p. Resistant mutants pdr11Δ and snq2Δ had
significantly lower vacuole formation than the WT after TcPR-10p exposure. Real
time microscopy was used to observe vacuole formation after exposure to TcPR-10p
in WT and snq2Δ and in M. perniciosa hyphae. Putative M. perniciosa autophagy
gene MpATG8 was introduced into yeast mutant atg8Δ and tested for heterologous
expression via phenotypic complementation of TcPR-10p sensitivity. Formation of
oxygen radicals (ROS) after exposure to TcPR-10p was observed using fluorescence
microscopy with dihydroethidium-stained cells. WT and mutant atg8Δ transformed
with a single-copy vector containing MpATG8 gene showed similar resistance to
TcPR-10p and similar formation of ROS, while mutant atg8Δ was sensitive and
exhibited increased ROS accumulation. This suggests that the protein codified by
MpATG8 is functionally expressed in S. cerevisiae and protects against TcPR-10p
whereas mutant atg8Δ accumulates ROS under the same conditions. Our results
suggest (1) that TcPR-10 protein uses a cell penetration route similar to that of
sterols as suggested by the resistance of mutants pdr11Δ and snq2Δ; (2) that
vacuole accumulation can be taken as indication of intracellular TcPR-10p activity in
both yeast and M. perniciosa and (3) that a functional autophagic mechanism is
essential for the WT-resistance of yeast to TcPR-10p-induced oxidative damage.
64
AUTHOR SUMMARY
Moniliophthora perniciosa is a basidiomycete fungus that causes Witches’ broom disease of Theobroma cacao, responsible for heavy economic damage in several countries. A previous study showed that the antifungal protein TcPR-10p, isolated from cocoa, has in vitro and in vivo ribonuclease activity and inactivates survival of M. perniciosa and Saccharomyces cerevisiae. Here we report that the transport of TcPR-10p into the yeast cell is via the sterol route (transporter Pdr11p a member of the ATP-binding cassette) and that absence of Pdr11p induces TcPR-10p resistance in the yeast S. cerevisiae. We show that the increased susceptibility to TcPR-10p in S. cerevisiae and M. perniciosa is coupled with vacuole formation. The absence of the autophagic mechanism in the yeast atg8 mutant leads to a three-fold higher TcPR-10p sensitivity via increased formation of reactive oxygen species (ROS) in S. cerevisiae. The expression of a single copy of M. perniciosa MpATG8 restores low ROS levels and near WT survival. We can conclude that increased TcPR-10p exposure leads to increased intracellular oxidative stress. To our knowledge this is the first work to relate that antifungal activity of PR-10 is associated with induction of oxidative stress most probably contributing to cell death.
65
INTRODUCTION
Plants are subject to several types of biotic and abiotic stresses. Thus, they
have developed adaptive attack perception and defense manifestation mechanisms
that allow their survival at different stress levels [1-4]. Once stress is perceived at the
molecular level, plants unchain a complex cascade of events that subsequently
activate many defence responses, such as production of pathogenesis-related
proteins (PR). PR proteins play an important role in induction of resistance to
pathogens, and in some cases also of a constitutive resistance [5].
PR proteins are classified into 17 families according to their sequence and
biological activity [6,7]. Amongst them, the PR-10 family is highlighted as a group of
small acidic proteins found in mono- and dicotyledones that is in charge of the
intracellular defence process [8-10]. PR-10 family members have a highly conserved
glycine-rich motif called “p loop motif” (GXGGXGXXK; 47-55 amino acids) directly
related to their ribonuclease activity [11,12]. These PR-10p contain phosphorylation
sites characteristic of cAMP-dependent kinases [13]. The presence of 3 conserved
phosphorylation sites in PR-10p suggests a function as general RNase or possibly as
specific RNase against exogenous RNA [5,14,15]. Recently a DNAse activity of PR-
10p in rice [16] and grape was related [17] suggesting that PR-10 has a nuclease
activity and may be involved in the process of programmed cell death.
Witches’ broom disease of Theobroma cacao is caused by the hemibiotrophic
fungus Moniliophthora (formally Crinipellis) perniciosa. This phytopathogen
basidiomycete is responsible for heavy losses in Brazilian cacao production causing
serious socio-economic impact [18]. In order to better understand the processes
involved in this plant-pathogen interaction, Gesteira et al. [19] constructed two cDNA
libraries from cocoa cultivars resistant and susceptible to M. perniciosa and isolated
the gene coding for TcPR-10p (PR protein 10 of T. cacao). While Pungartnik et al.
[20] showed ribonuclease and antifungal activity of the heterologous expressed
protein TcPR-10p in M. perniciosa and Saccharomyces cerevisiae, Menezes et al.
[21] showed that the allergenic potential of TcPR-10p may be diminished or
abolished with the introduction of specific point mutations without loss of RNase
activity. TcPR-10p antifungal activity depends on its internalization that is energy and
temperature-dependent, suggesting an active importation into the fungal cell; also,
acute exposure of exponentially growing yeast cells revealed that TcPR-10p
66
resistance is enhanced in the Snq2 export permease-lacking mutant [20]. Yeast
Snq2p efflux pump activity is ATP-dependent, it is a multidrug transporter involved in
multidrug resistance and resistance to singlet oxygen species [22,23]. Snq2p is a
member of the ATP-binding cassette (ABC) family of proteins, a large group of
proteins that are conserved from bacteria to humans [24].
ABC transporters couple ATP hydrolysis to vectorial translocation of diverse
substrates across membranes [22], including metabolic products, lipids, sterols, and
drugs. The yeast S. cerevisiae contains several genes encoding ABC transporters
(For review see [22,25-27]), amongst them genes of the pleiotropic drug resistance
(PDR) subfamily that codify for ABC transporters and their regulators [28]. Yeast
mutants lacking PDR genes may be used as tools for the identification of routes of
elimination of many harmful substances, amongst them fungicides and antibiotics
[29,30]. Toxic agents entering the yeast cells induce or activate several fungal
mechanisms aimed at re-establishing cellular homeostasis and recycling damaged
macromolecules and organelles, mainly through the process of autophagy [31,32].
Both in vivo and in vitro ribonuclease activity of recombinant TcPR-10p were
observed on cacao and M. perniciosa RNA [20], which might be associated to the
reduction of fungal survival. In yeast, RNA degradation is tightly regulated and
numerous quality control mechanisms that target aberrant RNAs have been
identified. However, mechanisms that control the specificity of RNA degradation and
how RNA degradation processes interact with translation, RNA transport and other
cellular processes remain to be elucidated [33]. RNase activity may give origin to
aberrant transfer RNA (tRNAs), ribosomal RNA (rRNAs), as well as mRNAs; in yeast
tRNAs are imported into the vacuole or another membrane-bound compartment by
some type of autophagy-related process to be degraded by Rny1 (a vacuolar RNase
of the T(2) family), which relocalizes to the cytosol where it cleaves tRNAs upon
oxidative or stationary phase stress [34]; also, amongst the 3 types of degradation of
aberrant rRNA in yeast, one targets RNA to vacuoles by selective autophagy in
response to oxidative stress or entry into stationary phase. Ribosomes may also be
targeted for degradation in the vacuole by piecemeal micro-autophagy of the
nucleus, where regions of the latter are directled to the vacuole by invagination of the
nuclear envelope into the vacuolar lumen [35].
In this respect, autophagy is a conserved catabolic process that initially
involves the bulk or the selective engulfment of cytosolic components into double-
67
membrane vesicles and sequentially the transport of the sequestered cargo to the
lysosome/vacuole for degradation [36]. Usually triggered in eukaryotic cells to
overcome nutritional limitations or other stress conditions, e.g., reactive oxygen
species (ROS)-induced oxidative stress, autophagy involves the delivery of
cytoplasmic components into the mammalian lysosome or the plant and yeast
vacuole for degradation, thus generating an internal pool of recyclable molecules.
This process allows maintenance of cellular homeostasis [37,38].
Autophagy (or macro-autophagy) is a pathway for membrane transport to the
lysosome/vacuole [39]. Autophagy sequesters superfluous cytosolic components and
organelles into double-membraned autophagosomes, which finally fuse with
lysosomes/vacuoles for degradation of its contents. It occurs constitutively at basal
levels and can be selective in the presence of damaged organelles. During this
process the autophagosome, a double membrane-closed vesicle is formed, which
selectively engulfs degradation targets, such as cytotoxic protein aggregates and
damaged or surplus organelles [40]. The fusion of the outer membrane of
autophagosomes with lysosomal/vacuole membrane permits the degradation of the
engulfed material together with inner membrane. In this process, Atg8p is essential
for autophagosomal membrane formation and also in efficient incorporation of
degradation targets into autophagosomes in selective types of autophagy [41]. Atg8p
homologs have an additional unique feature that reside on the formed
autophagosomes, suggesting their possible involvement in autophagosomal
maturation, i.e., fusion between autophagosomes and endosomes/lysosomes [41]. S.
cerevisiae mutants lacking autophagy are defective in various aspects of
mitochondrial functions, which suggests a critical role of autophagy in mitochondrial
maintenance[42]. A yeast mutant lacking ATG8-encoded protein had lower rates of
oxygen consumption, reduced mitochondrial electron transport chain activities and a
lower mitochondrial membrane potential. In addition, some mutants defect in the
autophagic process generated higher levels of ROS [42]. Yeast mutants defective in
gene ATG8 are incapable of autophagy and vacuolar transport is decreased [43].
In this work, we report that TcPR-10p is transported via the yeast ABC
transporter Pdr11p and that its absence induces TcPR-10p resistance in S.
cerevisiae, that the susceptibility to TcPR-10p in S. cerevisiae and M. perniciosa is
coupled with vacuole formation and that the absence of the autophagic mechanism in
yeast mutant atg8Δ leads to a higher TcPR-10p sensitivity and increased ROS
68
formation, and that a single copy of ATG8 gene homolog from M. perniciosa can
restore WT-resistance and diminish the oxidative stress after TcPR-10p exposure.
69
RESULTS
Pungartnik et al. [20] have shown that S. cerevisiae Snq2p is involved in
TcPR-10p resistance. Therefore, other members of yeast PDR protein
subfamily mutants such as pdr5Δ, pdr10Δ, pdr11Δ, pdr12Δ, pdr15Δ, and yor1Δ
were screened for survival after exposure to TcPR-10p. Amongst these 6 ABC
transporter mutants, five had a WT-like phenotype and one mutant, pdr11Δ,
demonstrated hyper-resistance to TcPR-10p (Fig. 1). This indicates that there
exist at least two ABC transporters, Snq2p and Pdr11p, involved in yeast TcPR-
10p resistance.
Since intracellular TcPR-10p activity may give origin to different aberrant
RNAs, which may be targeted to vacuoles by selective autophagy, S. cerevisiae
LOG phase cells were exposed to TcPR-10p (3 µg/mL) and observed by optical
microscopy during 4 h (Fig. 2, WT and snq2Δ); also, both TcPR-10p resistant
mutants snq2Δ and pdr11Δ were stained with neutral red after 6 h exposure to
TcPR-10p (3 µg/mL) (Fig. 3A to 3D). Formation of vacuole-like structures was
observed in WT cells (Fig. 2B, 60 min), but not in yeast mutant snq2Δ exposed
up to 2 h to TcPR-10p (Fig. 2B). In fact, snq2Δ (Fig. 3B) and pdr11Δ (Fig. 3D)
TcPR-10p-exposed cells presented fewer neutral red-stained vacuoles when
compared to the isogenic WT (Fig. 3A and 3C). WT-like TcPR-10p-sensitive
mutants pdr5Δ, pdr10Δ, pdr12Δ, pdr15Δ, and yor1Δ showed a frequency of
stained vacuoles similar to that of the WT (data not shown).
Since TcPR-10p is a plant response due to successful M. perniciosa
infection, we also checked for formation of vacuole-like structures after TcPR-
10p exposure of the fungal hyphae (Fig. 4). The images obtained by real time
microscopy after TcPR-10p exposure (less than 30 minutes) showed a strong
increase in size and number of intracellular structures resembling vacuoles (Fig.
4B and film in supplementary material 4) as compared to images of non-treated
hyphae (Fig. 4A).
The observed TcPR-10p-induced formation of vacuoles in M. perniciosa
and yeast WT and other mutants with WT-like sensitivity led us to speculate that
we were witnessing induced autophagy, carried out by vacuole-like
autophagosomes [44,45]. If this were the case, the yeast autophagy-deficient
70
mutant atg8Δ should have a higher-than-WT sensitivity to TcPR-10p. Figure 5
clearly shows that it does.
Proteins involved in autophagy are highly conserved and the putative
MpATG8 gene encodes a protein with nearly identical structural features to the
yeast Atg8p (A. Pereira, unpublished). We cloned the M. perniciosa putative
gene MpATG8 and transferred it, contained in a single copy vector (pRS313),
via transformation into the yeast mutant atg8Δ and its isogenic WT. Information
on sequencing, amplification, cloning and expression of the gene is provided in
supplemented material (Supp. 1, 2, 3). Four yeast transformants with isogenic
background were constructed: AP01, (WT [pRS313]) and AP03 (atg8Δ
[pRS313]) harbouring the single-copy vector pRS313 whereas AP02 (WT
[pLBF3]) and AP04 (atg8Δ [pLBF3]) contained the MpATG8 gene-containing
single-copy vector. When these 4 yeast transformants were exposed to TcPR-
10p (3 µg/mL, up to 48 h, Fig. 5) only the non MpATG8-containing strain AP03,
the yeast mutant atg8Δ transformed with the empty vector, exhibited higher
sensitivity, as the non-transformed mutant atg8Δ (data not shown). The two WT
transformants AP01 and AP02 as well as the atg8Δ [pLBF3]) transformant
AP04 displayed WT-like TcPR-10p resistance.
Since RNAse activity of TcPR-10p in M. perniciosa has been shown [20]
and the increase of degraded RNA might generate intracellular oxidative stress
[46], we reasoned that this might be also the case in yeast and that cellular
death might be associated with ROS. As lack of autophagy renders the yeast
cells more TcPR-10p sensitive we therefore measured the oxidative stress
indicating superoxide anion levels by fluorescent labelling with dihydroethidium
(DHE) (Fig. 6). When DHE is introduced to a metabolic active cell that contains
ROS in the form of superoxide radicals it is oxidized to ethidium, which can be
identified by its intercalation into DNA. Thus, oxidative stress can be visualized
by treating yeast cells with DHE [47]. When the four yeast transformants were
challenged with TcPR-10p and then treated with DHE, the initially (0 h) low
incidence of superoxide radicals (ROS) increased after 48 h of TcPR-10p
exposure significantly in transformant AP03, i.e., in the yeast atg8Δ mutant
strain not containing a single copy of MpATG8 (AP03, Fig. 6C). However, a
single copy of the MpATG8 gene (AP04, Fig. 6D) was sufficient to reduce ROS
formation to that seen in WT transformants (AP01 and AP02) (Figs. 6A, B). The
71
same results were obtained challenging with 10-fold diluted TcPR-10p, however
with less intensity of fluorescence (data not shown).
DISCUSSION
1) Absence of ABC transporter Pdr11p leads to TcPR-10p resistance in the
yeast S. cerevisiae
Yeast ABC proteins are divided into subfamilies according to the
sequence similarity within their nucleotide-binding domain (NBD), which couples
nucleotide hydrolysis to substrate transport. Yeast transport mutant snq2Δ,
which is known to have higher-than-WT TcPR-10p resistance [20], belongs to
the ABC PDR5/ABCG transporter family [48]. Proteins of this group have similar
sequences within their NBDs, which tend to have partially overlapping
physiological and biochemical functions; although transporting unrelated
substrates, they are known to be ABC-efflux transmembrane proteins [30]. Of
the six further tested isogenic yeast ABC transport mutants yor1Δ, pdr5Δ,
pdr10Δ, pdr11Δ, pdr12Δ and pdr15Δ only pdr11Δ was found to be hyper-
resistant to TcPR-10p, i.e., had a similar phenotype as mutant snq2Δ [49], while
all other had a WT-like phenotype (Fig. 1).
Yeast gene SNQ2 codifies an efflux pump for multiple structurally
unrelated mutagens and drugs [24]. The absence of the export permease
function of Snq2p apparently hinders the export of a cellular component that,
when present in higher than usual amount, inhibits the cytotoxicity of TcPR-10p
in the respective mutant [20]. Pdr11p, an export permease involved in multiple
drug resistance, also mediates sterol uptake when sterol biosynthesis is
compromised. Sterols are an important class of lipids involved in several
functions, e.g., in signal transduction, vesicle formation, lateral aggregation
between proteins and lipids amongst others [50]. Studies of molecular
modelling, nuclear magnetic resonance and saturation transfer difference of the
structure of a PR-10 homolog, Betv1p, identified the existence of binding sites
for fatty acids, flavonoids, cytokines, emodin and sterols, suggesting that this
protein plays an import role in the storage and transport of biologically important
molecules, with activity in various tissues and conditions [51,52]. Binding of PR-
10p to sterols had already been demonstrated in plants: brassinosteroids,
72
hormones involved in the regulation of vegetal growth bind to Betv1p, allowing
their movement inside the plant [53]. Koisisten et al. [51] demonstrated that
dehydroergosterol can bind inside the hydrophobic cavity of Betv1p using Van
der Waals binding and that the majority of the amino acid residues involved in
this interaction are highly conserved. Indeed, TcPR-10p also shows the same
essential sequences of the Betv1p hydrophobic cavity [20]. Although Pdr5p and
Snq2p, Aus1p and Pdr11p of S. cerevisiae are known to transport steroids [54],
only the absence of Snq2p [20] and Pdr11p conferred hyper-resistance to
TcPR-10p (Fig. 1). That may be explained by the fact that only Pdr11p and
Snq2p and not the other two are regulated by transcription factors Pdr1p [55].
This points to an important role for activation of transcription factors by TcPR-
10p. We may, therefore, suggest that, owing to the same sterol affinity,
membrane bound TcPR-10p is dislocated from the membrane sterol by
membrane inserted Pdr11p; this could be the key element of uptake. A lower
transport of TcPR-10p in mutant pdr11Δ would lead to the observed resistance
phenotype.
Therefore, the observed resistance of the yeast cells to TcPR-10p seems
to be linked to at least two factors: 1) a non-functional membrane transport
mechanism (sterol import by both Pdr11p and Snq2p); and 2) the absence of
efflux pump activity for components deactivating TcPR-10p function.
2) Increased susceptibility to TcPR-10p in S. cerevisiae and M. perniciosa is coupled to vacuole formation
Higher amounts of structures resembling vacuoles (Fig. 2A) were
observed in the WT strain (sensitive to TcPR10p) in light microscopy as
compared to a likewise treated snq2Δ mutant (resistant to TcPR-10p, Fig. 2B).
Therefore, yeast cells under the same conditions were stained with neutral red
and representative photos are shown in Fig. 3 (A to D). Neutral red, a vital dye,
visible under white and bleaching resistant, is accumulated in the vacuolar
lumen [56]. This dye has been efficiently used for visualization of this organelle
in different fungi such as Botrytis cinerea [57], Magnaporthe grisea [58] and
Colletotrichum graminicola [59]; however, its use with M. perniciosa has not
been successful (data not shown). However, in this work M. perniciosa has
shown to be recalcitrant to staining of vacuoles with neutral red.
73
In yeast and basidiomycete fungi vacuoles are compartments involved
with degradation of cellular components, supply of ions and metabolites in order
to maintain cellular homeostasis of ions and pH [60,61]. After exposure to
TcPR-10p, both neutral red stained resistant snq2Δ and pdr11Δ mutants
exhibited only a few small vacuoles, indicating that the resistance to TcPR-10p
may be associated with the reduction of vacuole formation. All other tested
transport mutants had phenotypes similar to the WT (same sensitivity to TcPR-
10p and increased number of vacuole) as shown by neutral red staining (data
not shown).
In yeast, incorporation of sterol molecules into the plasma membrane is
not spontaneous - it needs the presence of Pdr11p and Aus1p [54]. The lack of
Pdr11p leads to TcPR-10p hyper-resistance (Fig. 1B) as well as to a reduced to
number of TcPR-10-induced vacuoles (Fig. 3D). As vacuoles are formed by
membranes and the presence of sterols is essential for the membrane
formation [62,63], the TcPR-10p hyper-resistance in both snq2Δ and pdr11Δ
mutants could be associated with the reduction of sterol capture from the
medium and, therefore, reduction of its availability for the formation of
membranes. The decreased TcPR-10p transport through the membrane,
specifically using the ABC type transport protein Pdr11p, leads to the observed
hyper-resistance phenotype (Fig. 1B), whereas in the WT strain and other
tested mutants of ABC membrane transporters, the active transport of TcPR-
10p leads to the sensitivity phenotype (this work Fig. 1 and Pungartnik et al.
[20].
TcPR-10p treated cells of M. perniciosa hyphae also displayed increased
formation of vacuole-like structures in Real Time Microscopy (Fig. 4),
resembling the features observed in TcPR-10p treated WT-like yeast cells (Fig.
1, 2 and 3). Thus, the observed TcPR-10p sensitivity of this basidiomycete
fungus seems to be associated with rapidly accumulated vacuole-like
structures. This process at the same TcPR-10p concentration was much more
rapid as compared to that in yeast (10 min in the former vs. 60 min in the latter).
The formation of vacuole-like structures in both organisms may be caused by
the induction of autophagy, a process already observed in M. perniciosa where
gene MpATG8 was up-regulated after exposure to DNA damaging 4NQO and
74
H2O2 (generating oxidative stress) when hyphae were grown in glucose and
during events that preceded basidiocarp formation [49].
3) Absence of autophagy in yeast mutant atg8Δ leads to higher TcPR-10p
sensitivity via increased ROS formation.
The autophagic route is well conserved in different fungi [64], therefore,
heterologous expression of the putative gene MpATG8 of M. perniciosa in yeast
S. cerevisiae was successfully accomplished (Fig. 4, Supp mat). MpATG8 was
cloned in a single copy plasmid pRS313 [65] and transformed into WT and
atg8Δ mutant strains of yeast, that were then exposed to TcPR-10p (3 µg/mL).
As expected, the atg8Δ mutant (AP03) was highly sensitive to TcPR-10p (Fig.
5). This may be explained by the non-functionality of the autophagic route, i.e.
low induction of vacuoles [autophagosomes] that led to accumulation of
degraded RNA and/or increased ROS formation, inducing cell death (lower
survival). The transformed atg8Δ mutant containing a single copy of the
MpATG8 gene (AP04) had WT-like response (AP01 and AP02). The restoration
of WT-like resistance phenotype indicates a probable functional expression of
MpAtg8p in yeast mutant atg8Δ.
The increase of degraded RNA might generate intracellular oxidative
stress [46]. Therefore we stained the transformants AP01, 02, 03 and 04 after
treatment with TcPR-10p (3 µg/mL; 0 and 48 h) with DHE and followed ROS
induction by fluorescence microscopy (Fig. 6). The formation of free radicals
could be observed in all tested transformants, but mainly in transformant AP03
(Fig. 6C). ROS act as signalling molecules in several processes, including
autophagy [42] and in high levels are harmful to the cells, leading to
programmed cellular death (PCD) [66]. Our results suggest that increased
exposure to TcPR-10p, leads to increased RNAse activity in the cell that in turn
induces increased intracellular oxidative stress. This can be proven with
transformant AP03 whose non-functional autophagic process allowed the
accumulation of intracellular ROS, in clear contrast to transformant AP04
(mutant atg8Δ containing gene MpATG8) that presented WT-like fluorescence
and, therefore, less oxidative stress. We may thus suggest that the oxidative
75
stressgenerated by increased TcPR-10p exposure can be diminished/lowered
by the restoration of the autophagic route.
Recent results by Cavalcante-Silva (2013) (unpublished data) on
proteomics of M. perniciosa after exposure to TcPR-10p indicate an over
expression of proteins involved in oxidative stress response (e.g. heat shock
protein HSS1, thiazole synthase, chaperone protein Dna K), cellular redox
homeostasis (malate dehydrogenase), protein biosynthesis (eukaryotic
translation initiation factor 5A-1) and autophagy (autophagy –related protein
18).
In summary, our results indicate that a second yeast ABC transporter,
Pdr11p (apart from Snq2p), is involved in the uptake of pathogenesis related
TcPR-10p, and that, once in the cell, this protein induces formation of vacuole-
like structures. Resistance to TcPR-10p in yeast is associated with low vacuole
formation. Increased formation of vacuole-like organelles in both WT yeast and
M. perniciosa after TcPR-10p exposure is associated with sensitivity to this
fungicide. Absence of autophagy in yeast leads to high TcPR-10p sensitivity,
which can be phenotypically complemented by transforming a yeast atg8Δ
mutant with a single copy of the MpATG8 gene. Restoration of WT resistance to
TcPR-10p and to reduced levels of ROS indicate that heterologous functional
expression of MpAtp8p restores autophagy. Our research gives a first indication
that intracellular TcPR-10p induces ROS and vacuoles and suggests that
autophagy plays an important role in survival of cells exposed to this protein.
Acknowledgments. Research supported by grants from CNPq and BNB. We
thank Acassia Leal for access to her cDNA library, Wagner Macena for
technical support constructing the plasmids and Marco Antonio Costa for using
the LEICA microscope. F.A.C. Silva held a CAPES doctoral fellowship
(Programa de Pós-Graduação em Genética e Biologia Molecular) and, A. C. F.
Pereira held a CNPq fellowship (Programa de Pós-Graduação em Biologia e
Biotecnologia de Microrganismos). C.P. held a DESI program fellowship at
CIRAD (La recherche agronomique pour le développement), Montpellier Rio
Imaging, France.
76
MATERIALS AND METHODS
Yeast strains, media, solutions and growth conditions
S. cerevisiae strains used in this work are listed in Table 1. Media,
solutions and buffers were prepared according to Burke et al. [67]. Strains were
routinely grown in rich liquid medium YEL (20 g/L glucose, 20 g/L peptone, 10
g/L yeast extract) under constant aeration in a gyratory shaker (New Brunswick,
G-76) for 2 to 3 days to stationary growth phase (STAT cells, density of
approximately 2 × 108/mL) or in solid medium YPD (YEL + 20 g/L agar) at 28
°C. Exponentially growing cells (LOG cells) were obtained by suspending a
colony in 100 μL saline (0.9% NaCl), which was spread on a YPD plate and
incubated overnight (16 – 18 h at 30 °C). The cells were collected, washed
three times (5 mL saline, 3 min, 5000 rpm) and resuspended to a final
concentration of 1 × 108 cells/mL. A culture was considered in LOG phase when
showing at least 30% of budding cells. To ascertain their respiratory
competence and for elimination of spontaneously accumulated petites, all
strains were pre-grown on solid YPG medium (YPD in which glucose was
replaced by 2% glycerol) before being used for experimentation.
Yeast survival after TcPR-10p exposure
S. cerevisiae was exposed to TcPR-10p in the following manner: yeast
LOG cells (OD660 between 1,2 to 2,4 x107/ mL) were treated in phosphate
buffer (50 mM; pH 7.4) containing 3 μg/mL TcPR-10p, at 25°C and samples
were collected up to 48 h at different exposure times. Samples were
appropriately diluted, plated on YPD agar and assayed for survival after 2 to 3
days of incubation at 28 °C. Graphs were generated by the GraphPad Prism®
program (GraphPad Software Incorporation. San Diego, CA); error bars
represent standard deviations of at least 3 independent experiments. Survival is
presented in a semi-log graph [68] for rapid estimation of dose reduction factor.
When not observable, error bars are minor or equal size of respective symbol.
77
M. perniciosa growth conditions
M. perniciosa strain ALF553 cultures were grown as described by Filho
et al. [69]. Dikaryotic cultures were grown in CPD (2% glucose, 2% peptone) in
liquid media, without agitation, at 25 °C for 5 to 7 days. M. perniciosa was
obtained from the fungal collection of UESC, originally kept at Comissão
Executiva do Plano da Lavoura Cacaueira.
Real time microscopy of TcPR-10p treated yeast and M. perniciosa
S. cerevisiae LOG cells of WT and snq2Δ mutant were placed in an
observation chamber (lifetechnology-Switzerland) and microscopically observed
during up to 4 h of TcPR-10p exposure (3 µg/mL). The same procedure was
used with dikaryotic strain 533 of M. perniciosa. The system of Real Time
Microscopy Richardson-Perkin Elmer [RTM 2.5 680 x 480 pixels, Sony 3CCD]
at CIRAD (La recherche agronomique pour le développement), Montpellier Rio
Imaging, France, was used. Cells were observed under an immersion oil 100 X
objective Fluotar (Numeral aperture 1,3). RTMicroscopy is based on black field
microscopy particularly suitable to observe living cells in a non invasive way
with a high spatial resolution (800 nm). A movie was recorded (Movie S1-
supplementary material) and pictures were extracted.
TcPR-10p induced vacuole formation
S. cerevisiae LOG cells of WT, snq2Δ and pdr11Δ mutants were
exposed to TcPR-10p for 0 and 6 h and then washed 3x with saline solution, re-
suspended in 100 µL of saline and incubated at 28 °C with Neutral Red (0.2
mg/mL) for 30 min in the dark. Vacuole formation was observed under a light
microscope (Olympus CX41) using 40x objectives in bright field.
Cloning of MpATG8
MpATG8 of M. perniciosa cDNA was identified from a previously
constructed mycelial cDNA library in the pDNR-LIB plasmid using DB SMART
Creator cDNA (Clontech) that had been derived from primordia and mature
basidiomata [70]. Molecular genetics methods were according to Ausubel et al.,
78
[71] and yeast methods according to Burke et al. [67]. MpATG8 putative gene
(supplemental material, 1) was amplified and ligated into plasmid pRS313 [65]
generating pLBF13 (supplemental material, 2). Yeast WT and atg8Δ mutant
were transformed with either empty pRS313 or pLBF3 plasmid, generating
transformants AP01 (WT [pRS313]); AP02 (WT [pLBF3]); AP03 (atg8Δ
[pRS313]) and AP04 (atg8Δ [pLBF3]). Yeast transformants were grown in
selective medium SC-His (1.7 g/L nitrogen base without amino acids, 5 g/L
ammonium sulphate, 20 g/L glucose, 0.04 g/L adenine hemisulfate, 0.06 g/L L-
leucine, 0.03 g/L L-lysine, 0.04 g/L L-tryptophan, 0.02 g/L uracil). Transformants
were checked for TcPR-10p resistance as described above.
Fluorescence assay of TcPR-10p induced ROS
LOG phase yeast transformants AP01, AP02, AP03 and AP04 were
treated with 3µg/mL TcPR-10p for 48 h. Stock solution (1 mg/mL) of the
fluorogenic probe dihydroethidium (DHE, SIGMA-ALDRICH®) was prepared by
dissolving it in dimethyl sulfoxide (Sigma, St. Louis, MO, USA). One mL of yeast
cells was stained by addition of 1 μL of stock solution and mixed by inversion,
incubated for 30 min at 28 oC, washed 3 times with saline, and re-suspended in
100 μL of saline. An aliquot was used to check oxidative/reductive stress of the
cells. Cytosolic DHE when oxidized by ROS (singlet oxygen, hydroxyl radicals,
superoxide, hydroperoxides and peroxides) yields ethidium, which intercalates
with a cell´s DNA and fluoresces brightly red (λ=605 nm) [47]. ROS induction
was observed by fluorescence microscopy DMRA2 (Leica®) attached with DHE
filter. Images were captured using 40x objective under bright field as well as
under fluorescent filters using the IM50 software (Leica®). Photos represent 5
samples from at least 3 independent experiments.
79
Figure Legends:
Figure 1. Survival of S. cerevisiae in LOG phase exposed to TcPR-10p (3
µg/mL) for 0, 1, 6 and 24 h: A) WT BY10000 (); pdr5Δ (X); pdr10Δ ();
pdr12Δ (); pdr15Δ (); yor1Δ (▲) and pdr11Δ ()
Figure 2. LOG phase cells of S. cerevisiae WT and snq2Δ mutant observed in
real time microscopy. A) non treated WT and snq2Δ kept for 2 and 4 h; B) cells
exposed to TcPR-10p (3 µg/mL): WT for 0, 60 and 90 min and snq2Δ for 0, 90
and 120 min. Bar: 10 µm.
Figure 3. Observation of vacuoles in LOG phase cells of S. cerevisiae exposed
to TcPR-10p (3 µg/mL) 0 and 6 h: A) WT BY10000, B) snq2Δ, C) WT BY10000
and D) pdr11Δ. Bar: 50 µm.
Figure 4. M. perniciosa hyphae observed in real time microscopy. A) Control;
B) Hyphae exposed to TcPR-10p (3 µg/mL).
Figure 5. Survival of yeast transformants exposed to TcPR-10p (3 µg/mL) for 0,
12, 24 and 48 h. () AP01 (WT [pRS313]) (WT [pLBF3]); () AP03
(atg8Δ [pRS313]) and () AP04 (atg8Δ [pLBF3]).
Figure 6. Production of ROS observed in epifluorescence photomicroscopy
after 0 and 48 h exposure to TcPR-10p (3 µg/mL). A) AP01 (WT [pRS313]), B)
AP02 (WT [pLBF3]), C) (atg8Δ [pRS313]) and D) AP04 (atg8Δ [pLBF3]). Bar:
50 µm.
80
Table 1 - Yeast strains and plasmid
Strains Genotype Source: Gene/protein function
BY (WT) MATα his3∆1 leu2∆0 lys2∆0 ura3∆0
EUROSCARF Wild type for membrane transporters and autophagy
yor1Δ Same as WT, YGR281w deleted
EUROSCARF Plasma membrane transporter involved in the tolerance to organic toxic anions
pdr5Δ Same as WT, YOR153w deleted
EUROSCARF Plasma membrane transporter involved in the resistance to multiple drugs
pdr10Δ Same as WT, YOR328w deleted
EUROSCARF Plasma membrane transporter involved in the resistance to multiple drugs
pdr11Δ Same as WT, YIL013c deleted
EUROSCARF Plasma membrane transporter involved in the absorption of sterol (ABC Transport)
pdr12Δ Same as WT, YPL058c deleted
EUROSCARF Plasma membrane transporter acting as a weak organic acid drawing pump
pdr15Δ Same as WT, YDR406w deleted
EUROSCARF Plasma membrane transporter
snq2Δ Same as WT, YDR011w deleted
EUROSCARF Multiple drugs efflux pump
atg8Δ Same as WT, YBL078C deleted
EUROSCARF
Expansion of the phagophore during autophagosome formation, influence the size of the autophagosome
AP01 (WT [pRS313])
Same as BY, containing pRS313
This work Wild type for membrane transporters and autophagy
AP02 (WT [pLBF3])
Same as AP01, containing putative ATG8 of M. perniciosa
This work -
AP03 (atg8Δ [pR313])
Same as atg8Δ, containing pRS313
This work Null atg8 mutations severely impair formation of autophagosomes
AP04 (atg8Δ [pLBF3])
Same as AP03, containing putative ATG8 of M. perniciosa
This work -
Plasmid name Relevant sequence
identification Source
pRS313 Single copy plasmid, HIS3 prototrophy Sikorsky & Hieter [65] -
pLBF3 Same as pRS3013 containing putative MpATG8
This work -
81
Figure 1
82
Figure 2
TcPR10 (3µg/mL) (minutes)
83
Figure 3
84
Figure 4
85
Figure 5
86
Figure 6
87
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SUPPLEMENTARY MATERIAL Supp. 1. Nucleotide and amino acid sequences of Moniliophthora perniciosa autophagy-related
protein 8 precursor (gi|189380199|and gi|189380200). Asterisk represents open reading frame
termination codon. MpATG8 pututive gene.
1 ATG AGG TCC AAA TTC AAG GAT GAG CAC CCC TTT GAG AAG CGC AAG GCT 48
M R S K F K D E H P F E K R K A
49 GAG GCG GAG CGT ATT CGA CAG AAG TAC CCA GAT CGT ATT CCT GTC ATC 97
E A E R I R Q K Y P D R I P V I
98 TGT GAG AAG GCG GAT AGA ACA GAT ATC CCC ACT ATT GAC AAG AAG AAG 114
C E K A D R T D I P T I D K K K
115 TAT TTG GTG CCC TCT GAT CTC ACT GTG GGC CAA TTC GTC TAT GTC ATT 131
Y L V P S D L T V G Q F V Y V I
132 CGC AAA CGG ATC AAG CTT GCA CCC GAG AAA GCC ATT TTT ATT TTC GTT 148
R K R I K L A P E K A I F I F V
149 GAC GAA GTA CTT CCG CCC ACA GCA GCT CTT ATG AGC GCT ATA TAC GAG 165
D E V L P P T A A L M S A I Y E
166 GAA CAC AAG GAC GAA GAC AAC TTT CTT TAC GTC AGC TAC TCG GGC GAG 182
E H K D E D N F L Y V S Y S G E
183 AAC ACG TTC GGG CAG GAA GGT TGG ATT GAG CTA CCG TCG GAT TCA TGA 199
N T F G Q E G W I E L P S D S *
Supp. 2. Molecular cloning of gene MpATG8 into vector pRS313. E= EcoR I; H= HIS3; A=
ARSH4; R= ampicillin gene
resistance. Generating
plasmid pLBF3.
Supp. 3 – PCR of yeast
transformants to confirm insertion of the
MpATG8 gene using specific primers. C+
amplification of cDNA of M. perniciosa. A =
AP01, B = AP02, D = AP03, C = AP04.
Movie Supp. 4 – WT 1h TcPR-10. mov: LOG phase cells of S. cerevisiae WT observed in real time
microscopy after exposure to TcPR-10p (3µg/mL) for 1h
Supp. 5 – MPTcPR-10. mov: M. perniciosa hyphae observed in real time microscopy after exposure to TcPR-10p (3 µg/mL) for 1h.
E
MpAtg8
E R A H
pRS313
91
VI. CONCLUSÕES GERAIS
A sensibilidade do fungo M. perniciosa à proteína PR10 (TcPR-10)
envolve proteínas de resposta a estresse e oxiduredutase indicando
possíveis formas de ação desta proteína antifúngica
TcPR-10 penetra na célula utilizando uma via similar a rota dos esteróis
conforme sugerido pela resistência dos mutantes pdr11Δ e snq2Δ
TcPR-10 induz formação de vacúolos e a rota autofágica é essencial
nos fungos M. perniciosa e S. cerevisiae
Primeiro relato de que TcPR-10 causa estresse oxidativo (ROS) em
fungos
