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 LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ 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 LNJ 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 Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD. Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast

“…Ribosome profiling and high-throughput genetic analyses has revealed hundreds of functional microproteins in human cells [ 76 , 77 ]. Documented functions for microproteins include phagocytosis [ 70 ], autophagy [ 71 ], actin-based motility [ 72 ], mRNA decapping [ 78 ], proteolytic processing [ 79 ], mitotic chromosome segregation [ 73 ], mitochondrial morphology control [ 76 ], plant hormone signaling [ 80 ], transcriptional control [ 81 ], and cancer cell survival [ 77 ], among many others [ 82 , 83 ]. Microproteins derived from noncanonical coding regions represent a substantial fraction of major histocompatibility complex (MHC) class I–associated tumor-specific antigens [ 84 , 85 ].…”

PAMP-triggered genetic reprogramming involves widespread alternative transcription initiation and an immediate transcription factor wave

“…The repression of the short AFP1 mRNA by flg22 treatment was confirmed by 5′-RACE ( Figure 4C ), and the unique appearance of Ribo-seq signal at the start codon of the short form in untreated tissues suggests that it can be translated into a microprotein comprising only the C-terminal domain, but neither the Ethylene-responsive binding factor-associated Amphiphilic Repression (EAR) nor Novel Interactor of JAZ (NINJA) domains with transcriptional co-repressor function ( Figure 4A ; Ohta et al, 2001 ; Pauwels et al, 2010 ). This AFP1-derived microprotein is nearly identical to the C-terminal domain of AFP2 (At1g13740; Figure 4D ) that was recently shown to be functionally equivalent to the naturally occurring microprotein LITTLE NINJA (LNJ) in monocots ( Hong et al, 2020 ). LNJ expression causes constitutive jasmonic acid (JA) responses, probably by interfering with homodimerization of the adaptor protein NINJA that is essential for linking core JA response regulators to the TOPLESS transcriptional co-repressor ( Pauwels et al, 2010 ; Hong et al, 2020 ).…”

“…C and E, Separation of PCR fragments obtained by 5′-RACE with primers specific for AFP1 (C) and PUS5 (E), organized as in Figure 3C . D, Sequence alignment of the microproteins resulting from translation of the naturally occurring short AFP1 mRNA isoform and of the AFP2 mRNA generated from the in-frame afp2 - cr6 deletion demonstrated experimentally to be biologically functional ( Hong et al, 2020 ). The NINJA-C domain is indicated in orange.…”

Controlling flowering of Medicago sativa (alfalfa) by inducing dominant mutations

“…The synthetic microProtein can interact and thereby inhibit these related proteins in a dominant-negative fashion (Figure 1). This has been shown in Arabidopsis, where parts of the coding sequence of the AFP2 gene that encodes a NINJA-domain protein were deleted by using a (CRISPR)/Cas-9 approach (Hong et al, 2020). NINJA proteins function as negative regulators of jasmonic acid (JA) responses.…”

“…NINJA proteins function as negative regulators of jasmonic acid (JA) responses. The NINJA-related microProtein, LITTLE NINJA (LNJ), was first discovered in Brachypodium as a factor affecting plant size and bushiness by interacting with NINJA and thus changing its jasmonic acid regulation (Hong et al, 2020). These findings show that engineering microProteins from individual genes is a possibility that has the potential to establish novel regulatory feedback loops.…”