Natural variation reveals key amino acids in a downy mildew effector that alters recognition specificity by an Arabidopsis resistance gene

Abstract

RPP13, a member of the cytoplasmic class of disease resistance genes, encodes one of the most variable Arabidopsis proteins so far identified. This variability is matched in ATR13, the protein from the oomycete downy mildew pathogen Hyaloperonospora parasitica recognized by RPP13, suggesting that these proteins are involved in tight reciprocal coevolution. ATR13 exhibits five domains: an N-terminal signal peptide, an RXLR motif, a heptad leucine/isoleucine repeat, an 11-amino-acid repeated sequence and a C-terminal domain. We show that the conserved RXLR-containing domain is dispensable for ATR13-mediated recognition, consistent with its role in transport into the plant cytoplasm. Sequencing ATR13 from 16 isolates of H. parasitica revealed high levels of amino acid diversity across the entire protein. The leucines/isoleucines of the heptad leucine repeat were conserved, and mutation of particular leucine or isoleucine residues altered recognition by RPP13. Natural variation has not exploited this route to detection avoidance, suggesting a key role of this domain in pathogenicity. The extensive variation in the 11-amino-acid repeat units did not affect RPP13 recognition. Domain swap analysis showed that recognition specificity lay in the C-terminal domain of ATR13. Variation analyses combined with functional assays allowed the identification of four amino acid positions that may play a role in recognition specificity. Site-directed mutagenesis confirmed that a threonine residue is absolutely required for RPP13 recognition and that recognition can be modulated by the presence of either an arginine or glutamic acid at other sites. Mutations in these three amino acids had no effect on the interaction of ATR13 with a resistance gene unlinked to RPP13, consistent with their critical role in determining RPP13-Nd recognition specificity.

Figure 1

Amino acid alignment and domains of ATR13 proteins encoded by 18 alleles from 16 isolates of Hyaloperonospora parasitica. Dots indicate synonymous amino acids to ATR13‐Maks9. Dashes indicate amino acids not present in variant form. The RXLR motif is underlined. Leucines/isoleucines in heptad repeat region are indicated by asterisks. Alleles whose encoded protein is above the black line are recognized by RPP13‐Nd in the biolistic assay and those below the line are not (# ATR13‐Goco1‐B is identical to ATR13‐Goco1‐A with one synonymous difference between ATR13‐Goco alleles, but not tested in the biolistic assay). In the C‐terminal region amino acids indicated by asterisks are those predicted to be key in the recognition response by RPP13‐Nd.

Figure 1

Amino acid alignment and domains of ATR13 proteins encoded by 18 alleles from 16 isolates of Hyaloperonospora parasitica. Dots indicate synonymous amino acids to ATR13‐Maks9. Dashes indicate amino acids not present in variant form. The RXLR motif is underlined. Leucines/isoleucines in heptad repeat region are indicated by asterisks. Alleles whose encoded protein is above the black line are recognized by RPP13‐Nd in the biolistic assay and those below the line are not (# ATR13‐Goco1‐B is identical to ATR13‐Goco1‐A with one synonymous difference between ATR13‐Goco alleles, but not tested in the biolistic assay). In the C‐terminal region amino acids indicated by asterisks are those predicted to be key in the recognition response by RPP13‐Nd.

Figure 2

ATR13 domain swap constructs and their recognition response by RPP13‐Nd in the biolistic assay. Region A and Region B are the two domains of the C‐terminal region and amino acids shown are the 11 amino acids that differ between ATR13‐Maks9 and ATR13‐Emoy2. Photographs (representative images) show the three distinct recognition response phenotypes in Col‐5 and Col‐5::RPP13‐Nd; no response results in 300–1000 blue‐stained cells per leaf, a full response results in fewer than ten per leaf and an intermediate response results in 50–150 per leaf.

Figure 3

C‐terminal regions of Emoy/Maks and Maks/Emoy domain swaps and mutants of domain swaps and their recognition responses by RPP13‐Nd in the biolistic assay. Grey shaded boxes indicate sequence from the ATR13‐Emoy2 allele; unshaded boxes indicate sequence from the ATR13‐Maks9 allele.

Figure 4

(A) C‐terminal region of mutant forms of ATR13‐Emoy2 and their recognition response by RPP13‐Nd in the biolistic assay. (B) C‐terminal region of ATR13‐Maks9, ATR13‐Wela3 and mutants of ATR13‐Wela3 and their recognition response by RPP13‐Nd. All differing amino acids between ATR13‐Maks9 and ATR13‐Wela3 are shown in the wild‐type constructs. Grey shaded boxes indicate sequence from ATR13‐Wela3; unshaded boxes indicate sequence from the ATR13‐Maks9 allele.

Figure 5

Recognition response of ATR13‐Wela3 mutants by UKID71 and Col‐5 in the biolistic assay.