Identificação e caracterização de proteinas expressas no espaço intercelular de folhas de Theobroma cacao na interação com Moniliophthora perniciosa / Identification and characterization of proteins expressed in the intercellular space of leves from Theobroma cacao infected by Moniliophthora perniciosa

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The invasion of the plant apoplast is a critical phase of the infection for numerous pathogens, including the fungus M. perniciosa (Mp) that remains in the intercellular space at the parasitic stage until about 60 days. Thus, the understanding of plant defenses induced in the apoplasm and the response mechanisms that the pathogens used for bypassing these defenses is essential to explain the plant-pathogen interaction. To establish a routine procedure for proteomic and biochemical analysis of cacao tissues, three new protocols were developed: one for apoplastic washing fluid (AWF) extraction, and two for protein extraction – under denaturing and non-denaturing conditions. The inhibitory effect of AWF from resistant cacao (TSH1188) and susceptible one (Catongo) on fungus basidiospores was analyzed, as well as chitinase, glucanase and peroxidase enzyme activities. The 2-DE of AWF protein profiles of healthy and infected TSH1188 were analyzed. Four cystatins, cysteine protease inhibitors, identified in cDNA library from T. cacao-Mp interaction were characterized. The first described method allows a quick and easy collection of AWF – using infiltration –centrifugation procedure – that is representative of its composition in intact leaves according to the little symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the non-denaturing protein extraction and subsequent enzymatic activity detection. AWF analyses revealed an enzymatic activity level and 2-DE profile that together with the inhibitory effect of AWF, suggest that during the initial stage of the T. cacao-Mp interaction, important mechanisms of defense are activated in the TSH1188 apoplast and repressed in Catongo. The peroxidase activity induction three days after TSH1188 inoculation, associated with literature information, reinforces the hypothesis of occurrence of an oxidative burst in the early stage of the interaction. These data suggest that the basidiospore germination test in AWF may be used for selection of cacao genotypes resistant to Mp, but the phyto-sanitary conditions of plants should be considered previously to the test. Cacao cystatin cDNA named TcCYS1, TcCYS2, TcCYS3 and TcCYS4 encode proteins with 209, 127, 124 and 205 amino acid residues, respectively. The corresponding His-tagged recombinant proteins were expressed in E. coli and showed inhibitory activity against Mp. TcCYS3 and TcCYS4-CNBr-sepharose immobilized captured proteases from Mp culture media. The Ki for papain with the colorimetric substrate BApNA was 203.2, 220.7, 152.4 and 158.9 for recombinant TcCYS1, 2, 3 and 4, respectively. Polyclonal antibodies raised against the recombinant TcCYS4 detected an endogenous protein more abundant in young tissues comparatively with mature tissues from cacao. A ~85 kDa cacao multicystatin induced by Mp inoculation, MpNEP (necrosis and ethylene-inducing proteins) and Mp culture supernatant infiltration was detected by anti-TcCYS4 antibodies in cacao meristems.

ASSUNTO(S)

moniliophthora perniciosa plant proteins plantas - proteinas theobroma

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