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

Abstract

ABSTRACT. 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. CO 2 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.

Identification and Characterization of Nep1-Like Proteins From the Grapevine Downy Mildew Pathogen Plasmopara viticola

“…Cystatins on the other hand can modulate cysteine proteases. TcCYS4 overexpression mutants of N. benthamiana showed clearly reduced sensitivity to necrosis formation by the transient expression of MpNEP2 (Santana et al, 2014). Nevertheless, transient PiNPP1.1 expression in V. vinifera leads to the induction of the sugar transporter VvSWEET4, suggesting a recognition of this NLP in V. vinifera (Chong et al, 2014).…”

“…Since P. viticola depends on a living host, one may assume the possibility of a mechanism for the detoxification of NLPs in these plants. One way of necrosis suppression was described in cacao plants (Theobroma cacao) by the action of the plant-derived cystatin TcCYS4 (Santana et al, 2014). The NLP MpNEP2 from Moniliophthora perniciosa activates cysteine proteases in cacao plants and induces hypersensitive cell death.…”

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

“…perniciosa [ 15 ]. By inducing the production of ethylene in the plant, this fungal protein induces the premature senescence process and triggers all cell death mechanisms in the plant [ 18 , 52 ]. There is a series of bands in the gel immunodetected by antiserum against TcCYSPR04, suggesting, mainly 72 hours after the interaction MpNEP-plant, the participation of various isoforms of cysteine-proteases in physiological events probable related to necrosis events triggered by MpNEP.…”

“…For this, tobacco leaves maintained under aseptic conditions were sprayed with 1.4 μM of recombinant NEP from M . perniciosa (MpNEP) [ 18 ] and collected at 24, 36, 40, 60 and 72 hours after spraying. As experimental control were used tobacco leaves maintained in the same conditions, but it not treated with MpNEP.…”

“…The TcCYS04 accumulation was also investigated in tobacco leaves treated with recombinant ethylene-inducing protein from M . perniciosa (MpNEP) [ 17 , 18 ]. Recombinant sepharose-immobilized cacao cystatin captured an active PLCP from cacao tissue, whose was confirmed by mass spectrometry as TcCYSPR04.…”