miR398b and AtC2GnT form a negative feedback loop to regulate Arabidopsis thaliana resistance against Phytophthora parasitica

Oomycetes are diploid eukaryotic microorganisms that seriously threaten sustainable crop production. MicroRNAs (miRNAs) and corresponding natural antisense transcripts (NATs) are important regulators of multiple biological processes. However, little is known about their roles in plant immunity against oomycete pathogens. In this study, we report the identification and functional characterization of miR398b and its cis-NAT, the core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase gene (AtC2GnT), in plant immunity. Gain- and loss-of-function assays revealed that miR398b mediates Arabidopsis thaliana susceptibility to Phytophthora parasitica by targeting Cu/Zn-Superoxidase Dismutase1 (CSD1) and CSD2, leading to suppressed expression of CSD1 and CSD2 and decreased plant disease resistance. We further showed that AtC2GnT transcripts could inhibit the miR398b-CSDs module via inhibition of pri-miR398b expression, leading to elevated plant resistance to P. parasitica. Furthermore, quantitative reverse transcription PCR, RNA ligase-mediated 5 '-amplification of cDNA ends (RLM-5 ' RACE), and transient expression assays indicated that miR398b suppresses the expression of AtC2GnT. We generated AtC2GnT-silenced A. thaliana plants by CRISPR/Cas9 or RNA interference methods, and the Nicotiana benthamiana NbC2GnT-silenced plants by virus-induced gene silencing. Pathogenicity assays showed that the C2GnT-silenced plants were more susceptible, while AtC2GnT-overexpressing plants exhibited elevated resistance to P. parasitica. AtC2GnT encodes a Golgi-localized protein, and transient expression of AtC2GnT enhanced N. benthamiana resistance to Phytophthora pathogens. Taken together, our results revealed a positive role of AtC2GnT and a negative regulatory loop formed by miR398b and AtC2GnT in regulating plant resistance to P. parasitica.