Genome-wide analysis of NDR1/HIN1-like genes in pepper (Capsicum annuum L.) and functional characterization of CaNHL4 under biotic and abiotic stresses

Abstract

Plant NDR1/HIN1-like (<i>NHL</i>) genes play an important role in triggering plant defenses in response to biotic stresses. In this study, we performed a genome-wide identification of the <i>NHL</i> genes in pepper (<i>Capsicum annuum</i> L.) and characterized the functional roles of these <i>CaNHL</i> genes in response to abiotic stresses and infection by different pathogens. Phylogenetic analysis revealed that <i>CaNHL</i>s can be classified into five distinct subgroups, with each group containing generic and specific motifs. Regulatory element analysis showed that the majority of the promoter regions of the identified <i>CaNHL</i>s contain jasmonic acid (JA)-responsive and salicylic acid (SA)-responsive elements, and transcriptomic analysis revealed that <i>CaNHL</i> genes are expressed in all the examined tissues of pepper. The <i>CaNHL1</i>, <i>CaNHL4</i>, <i>CaNHL6</i>, <i>CaNHL10</i>, <i>CaNHL11</i>, and <i>CaNHL12</i> genes were significantly upregulated under abiotic stress as well as in response to different pathogens, such as TMV, <i>Phytophthora capsici</i> and <i>Pseudomonas syringae</i>. In addition, we found that CaNHL4 localizes to the plasma membrane. <i>CaNHL4</i>-silenced pepper plants display significantly increased susceptibility to TMV, <i>Phytophthora capsici</i> and <i>Pseudomonas syringae</i>, exhibiting reduced expression of JA-related and SA-related genes and reduced ROS production. However, transient overexpression of <i>CaNHL4</i> in pepper increases the expression of JA-related and SA-related genes, enhances the accumulation of ROS, and inhibits the infection of these three pathogens. Collectively, for the first time, we identified the <i>NHL</i> genes in pepper and demonstrated that <i>CaNHL4</i> is involved in the production of ROS and that it also regulates the expression of JA-related and SA-related genes in response to different pathogens, suggesting that members of the CaNHL family play an essential role in the disease resistance of pepper.