A CaCDPK29-CaWRKY27b module promotes CaWRKY40-mediated thermotolerance and immunity to Ralstonia solanacearum in pepper.

CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high-temperature, high-humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood. Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK-containing WRKY protein CaWRKY27b, the calcium-dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH. Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W-boxes in the promoters of immunity- and thermotolerance-related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W-box binding ability. Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH.

Solanaceous plants switch to cytokinin-mediated immunity against Ralstonia solanacearum under high temperature and high humidity.

Plant diseases generally tend to be more serious under conditions of high temperature and high humidity (HTHH) than under ambient temperature, but plant immunity against pathogen attacks under HTHH remains elusive. Herein, we used pepper as an example to study how Solanaceae cope with Ralstonia solanacearum infection (RSI) under HTHH by performing RNA-seq combined with the reverse genetic method. The result showed that immunities mediated by salicylic acid (SA) and jasmonic acid (JA) in pepper roots were activated by RSI under ambient temperature. However, upon RSI under HTHH, JA signalling was blocked and SA signalling was activated early but its duration was greatly shortened in pepper roots, instead, expression of CaIPT5 and Glutathione S-transferase encoding genes, as well as endogenous content of trans-Zeatin, were enhanced. In addition, by silencing in pepper plants and overexpression in Nicotiana benthamiana, CaIPT5 was found to act positively in the immune response to RSI under HTHH in a way related to CaPRP1 and CaMgst3. Furthermore, the susceptibility of pepper, tomato and tobacco to RSI under HTHH was significantly reduced by exogenously applied tZ, but not by either SA or MeJA. All these data collectively suggest that pepper employs cytokinin-mediated immunity to cope with RSI under HTHH.

Pepper NAC-type transcription factor NAC2c balances the trade-off between growth and defense responses.

Plant responses to pathogen attacks and high-temperature stress (HTS) are distinct in nature but generally share several signaling components. How plants produce specific responses through these common signaling intermediates remains elusive. With the help of reverse-genetics approaches, we describe here the mechanism underlying trade-offs in pepper (Capsicum annuum) between growth, immunity, and thermotolerance. The NAC-type transcription factor CaNAC2c was induced by HTS and Ralstonia solanacearum infection (RSI). CaNAC2c-inhibited pepper growth, promoted immunity against RSI by activating jasmonate-mediated immunity and H2O2 accumulation, and promoted HTS responses by activating Heat shock factor A5 (CaHSFA5) transcription and blocking H2O2 accumulation. We show that CaNAC2c physically interacts with CaHSP70 and CaNAC029 in a context-specific manner. Upon HTS, CaNAC2c-CaHSP70 interaction in the nucleus protected CaNAC2c from degradation and resulted in the activation of thermotolerance by increasing CaNAC2c binding and transcriptional activation of its target promoters. CaNAC2c did not induce immunity-related genes under HTS, likely due to the degradation of CaNAC029 by the 26S proteasome. Upon RSI, CaNAC2c interacted with CaNAC029 in the nucleus and activated jasmonate-mediated immunity but was prevented from activating thermotolerance-related genes. In non-stressed plants, CaNAC2c was tethered outside the nucleus by interaction with CaHSP70, and thus was unable to activate either immunity or thermotolerance. Our results indicate that pepper growth, immunity, and thermotolerance are coordinately and tightly regulated by CaNAC2c via its inducible expression and differential interaction with CaHSP70 and CaNAC029.