Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

Abstract

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector-triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc-finger-like structure with a novel interleaved zinc-binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P-mediated recognition. The first zinc-coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid-binding and chromatin-associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non-conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.

Evolution of Disease Defense Genes and Their Regulators in Plants

“…In barley, the powdery mildew caused by Blumeria graminis , can be resistant by CNL type of genes including MLA10 [121], MLA1 [122], and MLA13 [123]. In Linum usitatissimum , flax rust caused by Melampsora lini (Fungal), can be resistant by TNL type of genes including P2 [124], L6 [125], M [126], L [127], L1-L11 [128,129], P [129,130], and P1 [124]. One disease resistance gene can also confer plants resistant to several plant diseases (Table 3).…”

Structural genomics applied to the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine knot and NTF2-like protein folds

“…The identification of plant targets of effectors associated with structure/function analyses of recombinant effectors can reveal how they interact with plant partners and how co-evolution with the host plants promotes the diversification of surface-exposed amino acids 1,11,26–29 . The avirulence proteins AvrL567, AvrM, and AvrP of the flax rust fungus Melampsora lini are the three effector structures described in rust fungi so far 10,11,30 .…”

EffectorP 3.0: prediction of apoplastic and cytoplasmic effectors in fungi and oomycetes

“…Whilst these effectors are incorrectly predicted with apoplastic as their most likely localization, they are still being predicted as cytoplasmic albeit with a lower probability. For example, AvrP123 is a cytoplasmic effector from the rust fungus Melampsora lini with 11 cysteines and a small size of 117 aas (9.4% cysteine content) and the cysteine residues are involved in zinc binding instead of disulfide bond formation (Zhang et al , 2018). EffectorP 3.0 predicts AvrP123 as cytoplasmic with probability 0.77 and apoplastic with probability 0.8, thus still recognizing the cytoplasmic signal despite the high cysteine content.…”