Functional Analysis of Barley MLA-triggered Disease Resistance to the Powdery Mildew Pathogen

More than 30 race-specific resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) map to the barley Mla locus. This exceptionally polymorphic locus harbors multiple members of three distantly related gene families, each encoding homologs of intracellular disease resistance (R) proteins. Previously isolated Mla1 and Mla6 R genes to Bgh encode highly sequence-related proteins (>90% identity) of approximately 105 kD that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leucine rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. A subset of Mla R genes are known to require for their function Rar1 and Sgt1. Intracellular RAR1 and SGT1 are known to physically associate and are thought to have co-chaperone-like activity. It was hypothesized that other Mla R gene specificities might be closely related to Mla1 and Mla6. A cosmid library was constructed from an Mla12-containing barley line and cosmids containing candidate Mla12 genes were isolated using molecular probes derived from Mla1. Race-specific resistance activity to an AvrMla12 containing Bgh isolate was detected following transient single cell expression of the Mla12 candidate gene in detached barley leaves. This and the identification of point mutations in susceptible mla12 mutants demonstrated that the candidate gene is Mla12. Over-expression of Mla12 in the single cell assay shifted the slow Mla12 resistance response in wild type plants to a rapid Mla1/Mla6-like resistance, in effect terminating fungal growth at the cell wall penetration stage before haustorium development. This indicates that the cognate AvrMla12 product must be secreted before the switch from surface to invasive fungal growth. Resistance mediated by Mla12 over-expression retained the requirement for Rar1. Single cell dsRNAi gene silencing experiments revealed a requirement of Mla12 resistance for Sgt1. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein regions required for the recognition of cognate Bgh effectors (encoded by AvrMla1 and AvrMla6). These regions comprise different but overlapping LRR regions and the CT part. This is consistent with the finding that the LRR and CT encoding parts of Mla1 and Mla6 exhibit evidence for diversifying selection. Unexpectedly, MLA chimeras that confer AvrMla6 recognition specificity exhibit markedly different dependence on Rar1. Furthermore, uncoupling of MLA6-specific resistance from RAR1 also uncoupled the resistance response from SGT1. These findings suggest that differences in the degree of RAR1 dependence of different MLA immune responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of presumptive resistance protein�containing recognition complexes. Previous analysis showed that loss-of-function mutations in both barley and Arabidopsis Rar1 severely impair accumulation of MLA and RPM1 NB-LRR proteins in healthy plants, respectively. In this study it was shown that co-expression of RAR1 and MLA1 or MLA6 in heterologous yeast is not sufficient to elevate MLA steady state levels. This suggests that RAR1 stabilizes MLA proteins indirectly, e.g. through other host factors that might be components of MLA containing recognition complex(es). Yeast 2-hybrid screenings were used to identify host proteins directly interacting with MLA R proteins. Several MLA interactors were identified using distinct or overlapping domains from MLA1 or MLA6 proteins. Amongst those is the SGT1 protein that is genetically required for the function of many NB-LRR proteins in diverse plant species. Other MLA interactors exhibit high sequence relatedness to carbonic anhydrase (CA), formin homology (FH) proteins, and a WRKY transcription factor. Interestingly, tobacco carbonic anhydrase was previously shown to be required for the activation of an efficient cell death response triggered upon race-specific recognition of the Pseudomonas syringae effector AVRPTO by the R gene Pto. The functional significance of the identified interactors for Mla-mediated race-specific resistance to Bgh remains to be validated, e.g. by dsRNAi experiments in the single cell gene expression assay.