TcCYS4, a cystatin from cocoa, reduces necrosis triggered by MpNEP2 in tobacco plants.

In Brazil, most cocoa bean production occurs in Southern Bahia. Witches' broom disease arrived in this area in 1989 and has since caused heavy losses in production. The disease is caused by the basidiomycete fungus Moniliophthora perniciosa, a hemibiotrophic fungus that produces the necrosis and ethylene-inducting protein (MpNEP2) during infection; this protein can activate cysteine proteases and induce programmed cell death. Cysteine proteases can be modulated by cystatin. In this study, we overexpressed TcCYS4, a cocoa cystatin, in tobacco plants and evaluated the effect on MpNEP2 in model plants. Tccys4 cDNA was cloned into the pCAMBIA 1390 vector and inserted into the tobacco plants via Agrobacterium tumefaciens. Transgene expression was analyzed by reverse transcription-quantitative PCR and Western blot analysis. Transcript and protein levels in Tcccys4:tobacco lines were 8.9- and 1.5-fold higher than in wild-type plants (wt). Tcccys4:tobacco lines showed no change in growth compared to wt plants. CO2 net assimilation (A) increased in Tcccys4:tobacco lines compared to wt plants. Only one line showed statistically significant stomatal conductance (gs) and transpiration rate (E) changes. MpNEP2 was infiltered into the foliar mesophyll of Tcccys4:tobacco lines and wt plants, and necrotic lesions were attenuated in lines highly expressing Tccys4. Our results suggest that cocoa cystatin TcCYS4 affects MpNEP2 activity related to the progression of programmed cell death in tobacco plants. This may occur through the action of cystatin to inhibit cysteine proteases activated by MpNEP2 in plant tissues. Further studies are necessary to examine cystatin in the Theobroma cacao-M. perniciosa pathosystem.