CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling.

Lei Shen,Sheng Yang,Wei Cheng,Cailing Liu,Shuilin He,Jiong Hu,Jiayu Wen,Shaoliang Mou,Lanping Shi,Yanyan Liu,Wei Shi,Jiaqi Liang,Zhiqin Liu,Hanyang Cai,Yang Wu,Jiazhi Li,Qian Tang,Li He,Yangwen Zhang,Tong Yang,Deyi Guan

doi:10.1038/srep22439

Abstract

CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.

Highlights

Calcium (Ca2+) is a ubiquitous second messenger involved in plant responses to abiotic and biotic stresses[6]
A pCaCDPK15-driven GUS reporter was expressed in pepper leaves by agroinfiltration, and GUS expression in pepper leaves was measured in response to Ralstonia solanacearum inoculation (RSI) and exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethephon (ETH)
The results showed that GUS expression was upregulated by RSI, SA, MeJA, ABA, and ETH, with different temporal expression patterns (Fig. 1b)

Summary

Introduction

Calcium (Ca2+) is a ubiquitous second messenger involved in plant responses to abiotic and biotic stresses[6]. WRKY members in different plant species have been implicated in responses to abiotic and biotic stresses, including drought[26,27], salt[26], cold[28], heat[27], pathogen[28] and virus[29] infection, and herbivore attack[30]. Our previous study reported that CaWRKY40 functioned as positive regulator in pepper’s response to Pseudomonas solanacearum attack and heat stress under high humidity[36], and CaWRKY40 was transcriptionally modulated by CaWRKY6, another member in the WRKY TF family in pepper[37]. The pepper CDPK protein designated as CaCDPK15 was found to regulate pepper response to Pseudomonas solanacearum attack by indirectly activating CaWRKY40 expression, and CaWRKY40 in turn activated transcriptional expression of CaCDPK15 by binding to the CaCDPK15 promoter, thereby establishing a positive-feedback loop

Methods

Results

Conclusion