# Wheat miR408 and miR159 Weaken the Virulence of Parastagonospora nodorum (Berk.) and Induce the Defense Response in Plants (Triticum aestivum L.) Against Pathogens

## TL;DR

Wheat miR408 and miR159 reduce the harmful effects of a fungal pathogen and boost wheat's defense against it.

## Contribution

First demonstration of wheat miRNAs penetrating fungal mycelium and suppressing pathogen virulence genes.

## Key findings

- Spraying siRNA408 and siRNA159 increased wheat resistance to Parastagonospora nodorum.
- siRNA159 and siRNA408 suppressed fungal virulence genes and transcription factors in the pathogen.
- Wheat defense response included hydrogen peroxide accumulation and activation of salicylic acid signaling.

## Abstract

The discovery of bidirectional microRNA transfer between two organisms during plant–microbe interactions and the ability of some fungal pathogens to absorb double-stranded RNA (dsRNA) or short interfering RNA (siRNA) from the environment provided an impetus for exploiting this mechanism in plant defense against pathogens. In this study, we investigated the role of conserved wheat microRNAs (miRNAs), miRNA408 and miRNA159, in inducing plant defense responses and suppressing the virulence of the phytopathogenic ascomycete fungus Parastagonospora nodorum, mediated by necrotrophic effectors (NEs) encoded by SnTox genes regulated by fungal transcription factors (TFs). The foliar spraying with in vitro synthesized siRNA408 and siRNA159 duplexes before inoculation with SnTox3-producing P. nodorum isolate increased wheat plant resistance to the SnB isolate and suppressed the pathogen growth and development. Most likely, silencing of the miRNA408 target genes TaCAT-2A, TaCAT-2B, and TaCLP1, and the miRNA159 target gene TaMYB65, led to the induction of a defense response of wheat plants against P. nodorum. This defense response was characterized by a decrease in the catalase activity, accumulation of hydrogen peroxide, activation of the expression of salicylic acid signaling pathway genes (TaWRKY13, TaPR1), and suppression of the expression of ethylene signaling pathway genes (TaEIN3, TaPR3). We demonstrated for the first time the ability of siRNA159 and siRNA408 to penetrate the mycelium of the pathogen P. nodorum and be involved in the cross-kingdom regulation of fungal genes to suppress the expression of some genes of NE (SnToxA, SnTox3) and fungal TFs (SnStuA). We predicted potential targets for wheat miRNA408 and miRNA159 in the P. nodorum transcriptome, making spray-induced gene silencing (SIGS) promising for use against this pathogen. These results provide valuable insights for studying the cross-kingdom transfer of plant miRNAs.