Functional analysis of RIN4 natural variants in association with plant disease resistance proteins

Abstract

Decades of studies on plant immune receptors, NLRs, and phytopathogen-secreted effectors have shown that plant guardee/decoy proteins are routinely required for the successful detection of effectors. Hence, investigation of the biological features of guardee/decoy has considerably facilitated the current understanding of how plants could efficiently recognize effectors. RIN4 is one of the most well-studied guardee/decoy proteins. RIN4 is targeted by multiple sequence-unrelated effectors and undergoes distinct types of biochemical modifications. Subsequently, the effector-induced RIN4 modification is specifically detected by corresponding NLRs. Thus, RIN4-mediated immune signaling is formed by a complex interaction between NLRs, RIN4, and effectors. Moreover, most land plant species encode one or more copies of RIN4 orthologs. Consistent with this, RIN4-mediated effector recognition is observed in different plant species, including Arabidopsis, soybean, tobacco, and apple. FB_MR5 is an apple NLR that requires RIN4 for the recognition of bacterial effector, AvrRpt2 secreted from Erwinia amylovora, a causal agent of dreadful plant disease, fire blight. Notably, transferring FB_MR5 to a susceptible cultivar of commercial apples provides effective resistance against E. amylovora strains carrying AvrRpt2, implying potential use in crop protection. However, the precise molecular mechanism of how RIN4 regulates NLR, particularly FB_MR5, remains vague. In Chapter 3, two polymorphic residues on RIN4, which were shown to be important to determine NLR regulation specificity (RIN4 specificity motif: RSM) were further investigated. We conducted a comparative analysis of 34 RIN4 natural variants originating from 28 different plant species and demonstrated the presence of four major types of RSM. Subsequently, we revealed that the RIN4-mediated NLR regulation specificity correlates with the type of RSM rather than overall RIN4 sequence homology. This suggests that the specificity between RIN4 and NLR is formed by a small set of natural polymorphisms on RIN4. In addition, we showed that one histidine residue corresponding to Arabidopsis RIN4 histidine at position 167 (AtRIN4 H167) was commonly required to regulate all NLRs tested in this study, implying the presence of conserved mode of association between RIN4 natural variants and NLRs in H167 dependent manner. Based on our findings, we could successfully modify RIN4 to expand the spectrum of NLR regulation specificity. Taken together, we propose that RIN4 natural variants possess great potential to regulate a wide array of NLRs when accompanied by small but delicate changes in specific residues. We speculate that this unique feature of RIN4 is a key to establishing novel effector recognition both in nature and artificial engineering. In Chapter 4, we report a novel natural variant of FB_MR5, MbMR5-Kor, isolated from the Korean wild apple species, Malus baccata. We found that MbMR5-Kor carries eight polymorphic residues compared to FB_MR5, among which one polymorphic change on the CC domain, A54V, significantly enhances the auto-activity when expressed in N. benthamiana. Based on mutants of MbMR5-Kor, we also showed that similar to RSM on RIN4, a small set of natural polymorphisms on NLR could alter compatibility with RIN4. Taken together, we propose that the compatibility between RIN4 and NLR can be easily changed by small polymorphisms on either side. This implies that in nature, the diverse pool of RIN4 homologs might function as potential candidates readily recruited by RIN4-interacting NLRs, such as FB_MR5, to make a novel form of compatibility.