Comparative analysis of RIN4, an evolutionarily-conserved regulator of disease resistance in plants

RPM1 interacting-protein 4 (RIN4) is targeted by at least three bacterial virulence effectors, AvrB, AvrRpm1 and AvrRpt2. Here I demonstrated that RIN4 was also proteolyzed by the effector AvrPto. In two solanaceous species this degradation was dependent on the resistance genes, Pto and Prf. Recognition of AvrPto by Pto activated a proteosomal pathway in both tomato and Nicotiana benthamiana that degrades RIN4. The site cleaved by AvrRpt2 was required for this degradation. All RIN4 homologs from multiple species contained domains required for cleavage by AvrRpt2 and interaction with AvrB. Although only one RIN4 homolog is present in Arabidopsis, there are multiple homologs in tomato and lettuce. All six RIN4 homologs were expressed in healthy tissue at moderate levels similar to the housekeeping gene, alpha-tublin. Transcriptional levels of two RIN4 genes but not a third were significantly enhanced in tomato while transcription of both RIN4 genes from lettuce remained constant after challenge with a pathogen or non-pathogen. Transgenic complementation tests in a rin4/rps2 Arabidopsis mutant demonstrated that RIN4 homologs from tomato and lettuce could partially restore resistance to P. syringae pv. tomato expressing avrB. Computer modeling predicted that RIN4 is a highly unstructured protein except for conserved regions that interact with AvrB and the two AvrRpt2 cleavage sites. Diversity in the other regions of the protein suggests that many of the phenotypes of RIN4 are dependent on these conserved regions. The effector proteins, AvrRpt2, AvrB, AvrRpm1PsmM2 and its ortholog AvrRpm1PssB728A, showed different activities in three different hosts. AvrRpt2 cleaved RIN4 homologs in tomato and promoted growth of a Pseudomonas pathogen in tomato plants. This virulence activity of AvrRpt2 in tomato required its cysteine protease activity. In N. benthamiana, AvrRpt2 cleaved RIN4 homologs and showed an avirulence phenotype. This avirulence phenotype also required its cysteine protease activity. AvrRpm1Pss B728A and AvrRpm1PsmM2 exhibited different phenotypes in modifying AtRIN4 and pathogenesis on Arabidopsis and N. benthamiana. In the absence of the RPM1 protein, AvrRpm1 PsmM2 promoted more pathogen growth than did AvrRpm1 PssB728A in Arabidopsis. Both AvrRpm1 homologs conferred partial avirulence in tomato. AvrB conferred avirulence in N. benthamiana which required interaction with RIN4 and binding of ADP.