Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Abstract

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.

Keywords: Magnaporthe oryzae; immunity; proteomics; rice; signalling peptide; small secreted protein; transcriptomics.

Figure 1

Strategy for identifying SSPs. Rice plants were infected by M. oryzae, samples were collected at 1, 2 and 3 days post‐inoculation and rice suspension cells were treated with chitin for 1, 3, 6 and 12 h. Before sample collection, suspension cells and medium were separated and the medium was passed through an anion‐exchange column. Protein samples were subjected to SDS‐PAGE and proteins smaller than 25 kDa were recovered for PAGE gel. RNA sequencing (RNA‐Seq) and mass spectrometry (MS) results were combined to identify small secreted proteins (SSPs) shorter than 250 aa and containing an N‐terminal signal peptide sequence.

Figure 2

SSPs identified by transcriptome analysis. (a) Number of differentially expressed genes (DEGs) (>2‐fold change, FDR < 0.05) in plants and suspension cells. Yellow and green bars indicate down‐regulated and up‐regulated genes, respectively. (b) Venn diagram showing the overlapping up‐regulated genes between plants and suspension cells. (c) Number of up‐regulated SSP genes (>2‐fold change, FDR < 0.05) in plants and suspension cells, respectively. (d) Venn diagram showing the overlapping up‐regulated SSP genes between plants and suspension cells. (e and f) cis‐regulatory elements identified in the promoter regions of the up‐regulated 79 SSP genes induced in rice plants infected with blast fungus (e) and the 90 up‐regulated SSP genes induced in suspension cells treated with chitin (f).

Figure 3

SSPs identified by proteome analysis. (a) Number of proteins identified by MS analysis in plants infected by M. oryzae and suspension cells and medium treated by chitin, respectively. Yellow and green bars indicate the results from trypsin digestion and double enzyme digestion combining trypsin and Asp‐N, respectively. (b) Venn diagram showing the number of overlapping proteins identified by trypsin digestion and double enzyme digestion. (c) Venn diagram showing the number of overlapping proteins identified from plants, suspension cells and medium. (d) Number of SSPs identified in plants, suspension cells and medium, respectively. Blue and orange bars indicate the results of trypsin digestion and double enzyme digestion combining trypsin and Asp‐N, respectively. (e) Venn diagram showing the number of overlapping SSPs identified by trypsin digestion and double enzyme digestion. (f) Venn diagram showing the number of overlapping SSPs identified in plants, suspension cells and medium.

Figure 4

Identified SSPs number.  (a) Venn diagram showing the number of overlapping SSPs identified in plants, suspension cells and medium after combining RNA‐Seq with MS results. (b) Venn diagram showing the number of overlapping SSPs identified by RNA‐Seq and MS.

Figure 5

Expression of OsRALFL s and OsPSK4 induced by PAMPs. OsRALFL7, OsRALFL8 and OsPKS4 were induced in suspension cells treated with chitin (a) or PGN (b). Expression levels of OsRALFL7, OsRALFL8 and OsPKS4 were quantified by qRT‐PCR with six biological replicates. Error bars indicate standard error (S.E). Statistically significant differences between treated suspension cells and mock control are depicted with asterisks (*, P < 0.05; **, P < 0.01 and ***, P < 0.001) according to the two‐tailed t‐test.

Figure 6

IRP is involved in rice immunity. (a) Protein alignment of IRP and its homologues. Predicted N‐terminal signal sequences are in a blue box. (b) IRP expression was induced in suspension cells treated with chitin or PGN. The expression level of IRP was quantified by qRT‐PCR with six biological replicates. Error bars indicate S.E. Statistically significant differences between the mock and chitin‐treated cells are depicted with asterisks (*, P < 0.05; **, P < 0.01 and ***, P < 0.001) according to the two‐tailed t‐test. (c) Effect of overexpression of IRP on the defence gene PAL1. Rice suspension cells overexpressing IRP were treated with chitin for 1 h. #1 and #2 are two independent lines of transgenic rice suspension cells overexpressing IRP . The expression levels of IRP and PAL1 were quantified by qRT‐PCR with four independent replicates. Error bars indicate S.E. Statistically significant differences between wild‐type (WT) and overexpression lines are depicted with asterisks (*, P < 0.05; **, P < 0.01; and ***, P < 0.001) according to the two‐tailed t‐test. (d) Overexpression of IRP induces MAPK activation. MAPK activation was detected in WT and IRP overexpression lines #1 and #2 suspension cells using anti‐phospho‐p44/42 MAPK antibody. CBB, Coomassie Brilliant Blue. Similar results were obtained in four independent experiments. (e) The protein abundance of IRP in the medium increased following chitin exposure. The 20‐aa peptide GEGWLEDGIGMVVDMLGELK of IRP was used as a spectral to match the MS/MS spectrum of the peptide acquired using the parallel reaction monitoring (PRM) method. The rice suspension cells were treated with chitin for 2 h in two independent experiments. The total intensity of the peptide in the medium was determined in the mock and chitin‐treated sample, respectively. Error bars indicate S.E. Statistically significant differences between the mock and chitin treatments are depicted with asterisks (***, P < 0.001) according to the two‐tailed t‐test.