N-Methyltransferase CaASHH3 Acts as a Positive Regulator of Immunity against Bacterial Pathogens in Pepper

Proteins with conserved SET domain play a critical role in plant immunity. However, the means of organization and functions of these proteins are unclear, particularly in non-model plants such as pepper (Capsicum annum L.). Herein, we functionally characterized CaASHH3, a member of class II (the ASH1 homologs H3K36) proteins in pepper immunity against Ralstonia solanacearum and Pseudomonas syringae pv tomato DC3000 (Pst DC3000). The CaASHH3 was localized in the nucleus, and its transcript levels were significantly enhanced by R. solanacearum inoculation (RSI) and exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and abscisic acid (ABA). Knockdown of CaASHH3 by virus-induced gene silencing (VIGS) compromised peppers' resistance to RSI. Furthermore, silencing of CaASHH3 impaired hypersensitive-response (HR)-like cell death response due to RSI and downregulated defense-associated marker genes, including CaPR1, CaNPR1, and CaABR1. The CaASHH3 protein was revealed to affect the promoters of CaNPR1, CaPR1, and CaHSP24. Transiently over-expression of CaASHH3 in pepper leaves elicited HR-like cell death and upregulated immunity-related marker genes. To further study the role of CaASHH3 in plant defense in vivo, CaASHH3 transgenic plants were generated in Arabidopsis. Overexpression of CaASHH3 in transgenic Arabidopsis thaliana enhanced innate immunity against Pst DC3000. Furthermore, CaASHH3 over-expressing transgenic A. thaliana plants exhibited upregulated transcriptional levels of immunity-associated marker genes, such as AtNPR1, AtPR1, and AtPR2. These results collectively confirm the role of CaASHH3 as a positive regulator of plant cell death and pepper immunity against bacterial pathogens, which is regulated by signaling synergistically mediated by SA, JA, ET, and ABA.