Convergent evolution of disease resistance genes

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TRENDS in Plant Science

Vol.9 No.7 July 2004

| Research Focus

Convergent evolution of disease resistance genes John M. McDowell Department of Plant Pathology, Physiology, and Weed Science, Fralin Biotechnology Center, Virginia Tech, Blacksburg, VA 24061-0346, USA

The resistance genes Rpg1-b in soybean and RPM1 in Arabidopsis recognize the same bacterial avirulence protein (AvrB). Recent map-based cloning of Rpg1-b has provided the first opportunity to compare functionally analogous R genes in distantly related species. Rpg1-b and RPM1 are not orthologs. Rather, these genes descended from distinct evolutionary lineages in which recognition of AvrB has probably evolved independently. This result, together with new insights into RPM1-mediated recognition of AvrB, provides an exciting opportunity to reconsider classical views on the evolution of pathogen recognition specificity. Bacterial plant pathogens have evolved large complements of ‘effector’ proteins that are exported into plant cells via Type III secretion [1]. Once inside the plant cell, effectors manipulate host regulatory networks to suppress defense responses or otherwise create a more comfortable niche. For their part, plants have evolved resistance (R) proteins that recognize specific effector proteins as harbingers of colonization [2,3]. R genes are commonly regarded as short lived, but several lines of evidence counter this view [4,5]. First, some R gene alleles have been maintained for thousands of years by balancing selection [6– 8]. Second, the tomato resistance genes Pto and Prf evolved their functionalities before Lycopersicon speciation [9,10]. Finally, R genes with recognition specificity for the same Avr protein can be found in distantly related species [11]. For example, Rpg1-b (GenBank Accession number AY452684) from soybean (Glycine max) and RPM1 (GenBank Accession number NM111584) from Arabidopsis thaliana recognize the bacterial effector AvrB (GenBank Accession number M21965). Did these functionally analogous R genes inherit a conserved recognition specificity from a shared ancestral gene, or did they independently evolve the same recognition specificity? The recent map-based cloning of Rpg1-b provides the first opportunity to address this question by comparing it to the well-studied RPM1 gene [11]. Distantly related plants recognize the same avirulence proteins The foundation for this study was laid more than a decade ago during the advent of bacterial avr gene cloning and functional characterization. avrB was cloned from a soybean strain of Pseudomonas syringae (bacterial blight) and used to define soybean RPG1 [12,13]. avrB was subsequently Corresponding author: John M. McDowell ([email protected]). Available online 5 June 2004 www.sciencedirect.com

transferred to a P. syringae strain that colonizes Arabidopsis; this transgenic strain was used to define genetically the Arabidopsis resistance locus RPG3 [14]. At the same time, avrRpm1 (GenBank Accession number NC002759) was cloned from a Brassica oleracea strain of P. syringae and used to define the Arabidopsis RPM1 locus [15], as well as functionally equivalent loci in soybean, bean and pea [16,17]. These two lines of research converged when mutational studies and map-based cloning revealed that Arabidopsis RPG3 and RPM1 are the same gene (now called RPM1) [18, 19]. In other words, RPM1 provides recognition specificity against avrB and avrRpm1. This dual specificity was surprising at the time because avrRpm1 and avrB share almost no sequence similarity and no common biochemical function could be discerned from their sequences. This enigma is addressed below. Map-based cloning of a soybean R gene To better understand the functional and evolutionary relationships of soybean Rpg1 and Arabidopsis RPM1, Tom Ashfield and colleagues in Roger Innes’ laboratory tackled the difficult task of cloning Rpg1 with a map-based approach. The first interesting discovery to emerge from fine-scale genetic mapping was that the soybean specificities for avrB and avrRpm1 are tightly linked but encoded by two distinct genes (named Rpg1-b and Rpg1-r, respectively), rather than by a single gene with dual specificity like RPM1 [17]. A cluster of NB-LRR (nucleotide bindingleucine rich repeat [2,3]) candidate genes was mapped to the Rpg1 locus [20]. One NB-LRR gene defined a restriction fragment length polymorphism found only in avrB-resistant soybean lines [11]. This gene was shown to contain a missense mutation in an ethyl methane sulfonate-induced, rpg1-b loss-of-function mutant. A transient complementation assay (adapted from Ref. [21]) was used to bypass the difficulties associated with soybean transformation and confirm that the wild-type Rpg1-b candidate gene restored resistance to susceptible lines. Rpg1-b and RPM1 belong to different evolutionary lineages With Rpg1-b in hand, its evolutionary relationship to RPM1 could be assessed by sequence comparisons and phylogenetic analysis. Like RPM1, Rpg1-b encodes an NB-LRR protein with a putative coiled-coil (CC) domain at the N-terminus. However, the genes share only 34% identity across the nucleotide-binding domain and are even more divergent in the LRR domain. More significantly, phylogenetic reconstructions placed these two genes in distinct evolutionary lineages (clades). Both of

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these clades contain genes from multiple monocot and dicot species, indicating that their ancestors diverged before the monocot –dicot divergence [22]. These groupings provide strong evidence that Rpg1-b and RPM1 are not orthologs (i.e. descended from the same lineage and separated by speciation). Rather, these genes belong to paralogous lineages that arose from gene duplications that occurred before the monocot– dicot split (Figure 1). Considering the degree of sequence divergence and evolutionary distance between Rpg1-b and RPM1, it is unlikely that their recognition specificities are shared by descent. To sum up, the RPM1 and Rpg1-b genes are homologous because they descended from a common ancestral CC-NB-LRR gene. However, the recognition specificities of these two genes appear to have evolved independently and therefore are analogous rather than homologous.

CC-NB-LRR duplications

Monocot–dicot split (>150 MYA) N1

N2

N3

N4

Horizontal transfer of avr genes?

RPG1-b

RPG1-m

RPM1

?

?

RIN4

AvrB

Avr Rpm1

AvrB and Avr Rpm1 TRENDS in Plant Science

Figure 1. Convergent evolution of the soybean Rpg1-b and Arabidopsis RPM1 resistance genes. A previous phylogenetic study [22] provided strong evidence that four coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) lineages (N1, N2, N3, N4) arose from gene duplications that pre-dated the monocot– dicot divergence. Tom Ashfield and colleagues’ subsequent analysis [11] places Rpg1-b in lineage N1 and RPM1 in lineage N2. Thus, RPM1 and Rpg1-b are related by descent but have not shared a common ancestor for .150 million years. Given the evolutionary distance between RPM1 and Rpg1-b lineages, Ashfield et al. hypothesize that they evolved avrB recognition independently, perhaps in response to horizontal transfer of avrB between Pseudomonas syringae strains that are specialized for legumes or brassicas. RPM1 has evolved to detect AvrB and AvrRpm1 indirectly, through their effect(s) on RIN4. By contrast, the Rpg1 cluster contains distinct R genes against AvrB and AvrRpm1. Rpg1-r has not been cloned, but is depicted in this figure as a paralog of Rpg1-b. It remains to be seen whether Rpg1-b and Rpg1-r interact directly with the corresponding Avr proteins, or indirectly through a third party such as soybean RIN4. www.sciencedirect.com

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Does Rpg1-b guard soybean RIN4? To understand fully how and why this apparently convergent evolution occurred, it is necessary to understand what the R proteins recognize. Long-standing evolutionary hypotheses, based on the presumption of receptor– ligand interactions between cognate R and Avr proteins, would predict that Rpg1 and RPM1 have independently evolved to bind AvrB directly. The limited sequence similarity between RPM1 and Rpg1 argues against this hypothesis, as does the aforementioned dual specificity of RPM1 for two sequence-unrelated Avr proteins. A possible resolution of this enigma has been postulated from recent studies of protein – protein interactions between RPM1, AvrB, AvrRpm1 and an RPM1-interacting protein called RIN4 (GenBank Accession number NM113411). RPM1 does not bind directly to AvrB or AvrRpm1. Rather, RPM1 associates with RIN4 and detects phosphorylation or other modifications of RIN4 that are effected (directly or indirectly) by AvrRpm1 and AvrB [23]. Another P. syringae effector (AvrRpt2, GenBank Accession number L11355, a putative cysteine protease [24]) triggers destabilization of RIN4 and RPM1 proteins, thereby suppressing RPM1-dependent resistance [25– 27]. Thus, RIN4 appears to be targeted by three distinct Avr or effector proteins. The interactions between RPM1, RIN4 and various Avr proteins are consistent with the guard model for R – Avr interactions. This model departs from classic receptor– ligand models by predicting that R proteins detect modifications of host proteins targeted by effectors, rather than the effectors themselves [2,28]. The exact function of RIN4 is unknown, but the available evidence indicates an important role in RPM1 signaling and/or basal defenses that limit the spread of virulent pathogens [25,26]. Thus, it is reasonable to hypothesize that RIN4 is a popular virulence target in legumes and brassicas, and that RPM1 and Rpg1-b have independently evolved to detect modification of RIN4. Ashfield et al. provided indirect support for this model by demonstrating that AvrRpt2 can suppress Rpg1-b resistance [11]. They also reported a tantalizing but inconsistent destabilization of soybean RIN4 protein levels in response to AvrRpt2 expression. However, AvrB expression did not produce the predicted RIN4 mobility shift (from phosphorylation) on western blots. At present, the issue of RIN4 involvement in Rpg1-b –AvrB interaction remains open. Cluster of comparative opportunities The publication by Ashfield et al. [11] is exciting because it provides the first evidence for convergent evolution of R genes and opens the door for additional comparative studies that focus on the broad applicability of the guard model and the implications for plant– pathogen coevolution. Did Rpg1-b evolve to bind AvrB directly, to guard soybean RIN4 or to guard another soybean protein targeted by AvrB (Figure 1)? The increasing experimental evidence for the guard model favors the hypotheses that Rpg1-b evolved to guard soybean RIN4 or to guard another soybean protein targeted by AvrB. However, certain R proteins appear capable of direct interaction with the corresponding Avr [3,29] so we cannot dismiss the possibility yet that Rpg1-b has evolved a mode of AvrB

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interaction distinct from that of RPM1. It is perhaps noteworthy that the strongest experimental support for the guard model [23,25,26,30] has come from studies of single-copy R genes that are evolving conservatively [6– 8]. Does the guard model apply to multi-copy gene clusters in which strong diversifying selection has produced functionally distinct R genes? Have these gene clusters diversified to bind emergent effectors, or to monitor newly ‘at-risk’ guardees? R genes against oomycetes, viruses and nematodes reside close to Rpg1-b, and this interval contains a large cluster of NB-LRR genes that are related but evolving rapidly [20,31,32]. Have these genes evolved to detect manipulation of the same or similar virulence targets by diverse pathogens? Also, recall that the Rpg1 cluster contains two specialized resistance genes for AvrB and AvrRpm1, rather than a single gene with dual specificity [11,17]. Do AvrB and AvrRpm1 target different host proteins in soybean that are independently guarded by Rpg1-b and Rpg1-r? Is the Rpg1-r gene a recently duplicated paralog of Rpg1-b? Are the bean and pea genes for avrRpm1 resistance orthologous to Rpg1-r [16]? It should be enlightening to extend comparisons to Rpg1-r and to genes such as Rsv1, which is tightly linked to Rpg1-b but provides resistance to soybean mosaic virus [31,33]. In spite of the recent development of important experimental resources for map-based cloning and functional genomics, soybean remains much less tractable than Arabidopsis and other model plants. However, Ashfield and colleagues have provided a nice demonstration of how experimental protocols [21] and basic insights [23,25,26] from Arabidopsis research can be used to open up an important experimental system for comparative studies, with potential economic benefits. Acknowledgements I am grateful to Elizabeth Grabau, Mohammad A. Saghai-Maroof, and Stacey Simon for insightful comments. Funding from the NIH-NIGMS and the USDA-NRICGP supports my laboratory.

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