Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling

Abstract

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of <i>LNJ</i> in <i>Arabidopsis</i> resulted in stunted plants that resembled the decuple <i>JAZ</i> (<i>jazD</i>) mutant. In fact, comparing the transcriptomes of transgenic <i>LNJ</i> overexpressor plants and <i>jazD</i> revealed a large overlap of deregulated genes, suggesting that ectopic <i>LNJ</i> expression altered JA signaling. Transgenic Brachypodium plants with elevated <i>LNJ</i> expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of <i>LNJ</i> in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in <i>Arabidopsis</i> by deleting parts of the coding sentence of the <i>AFP2</i> gene that encodes a NINJA-domain protein. These <i>afp2-crispr</i> mutants were also stunted in size and resembled <i>jazD</i> Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.