Argonaute4 Modulates Resistance to <i>Fusarium</i><i>brachygibbosum</i> Infection by Regulating Jasmonic Acid Signaling

Abstract

Argonautes (AGOs) associate with noncoding RNAs to regulate gene expression during development and stress adaptation. Their role in plant immunity against hemibiotrophic fungal infection remains poorly understood. Here, we explore the function of AGOs in the interaction of wild tobacco (<i>Nicotiana attenuata</i>) with a naturally occurring hemibiotrophic pathogen, <i>Fusarium brachygibbosum</i> Among all AGOs, only transcripts of <i>AGO4</i> were elicited after fungal infection. The disease progressed more rapidly in AGO4-silenced (irAGO4) plants than in wild type, and small RNA (smRNA) profiling revealed that 24-nucleotide smRNA accumulation was severely abrogated in irAGO4 plants. Unique microRNAs (miRNAs: 130 conserved and 208 novel, including 11 canonical miRNA sequence variants known as "isomiRs") were identified in infected plants; silencing of AGO4 strongly changed miRNA accumulation dynamics. Time-course studies revealed that infection increased accumulation of abscisic acid, jasmonates, and salicylic acid in wild type; in irAGO4 plants, infection accumulated lower jasmonate levels and lower transcripts of jasmonic acid (JA) biosynthesis genes. Treating irAGO4 plants with JA, methyl jasmonate, or cis-(+)-12-oxo-phytodienoic acid restored wild-type levels of resistance. Silencing expression of RNA-directed RNA polymerases <i>RdR1</i> and <i>RdR2</i> (but not <i>RdR3</i>) and <i>Dicer</i>-<i>like3</i> (<i>DCL3</i>, but not <i>DCL2</i> or <i>DCL4</i>) increased susceptibility to <i>F</i> <i>brachygibbosum</i> The relevance of <i>AGO4</i>, <i>RdR1</i>, <i>RdR2</i>, and <i>DCL3</i> in a natural setting was revealed when plants individually silenced in their expression (and their binary combinations) were planted in a diseased field plot in the Great Basin Desert of Utah. These plants were more susceptible to infection and accumulated lower JA levels than wild type. We infer that AGO4-dependent smRNAs play a central role in modulating JA biogenesis and signaling during hemibiotrophic fungal infections.