Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening
Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions. Overall design: Reference: Wang et al., Plant Biotechnol J. 2020 Feb;18(2):415-428.  doi: 10.1111/pbi.13208.  We present a refined framework for isolating secreted signaling peptides involved in rice immunity by combining transcriptome and proteome analyses with functional assays using rice suspension cells. Three different materials were analyzed: 1) rice plants infected with rice blast fungus, 2) rice suspension cells treated with fungal elicitor chitin, and 3) media from the rice suspension samples. Two omic approaches were performed to identify differentially expressed genes identified by transcriptome and proteins detected exclusively in rice blast- and/or chitin-treated samples by proteome.