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

Abstract

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

Remote homology clustering identifies lowly conserved families of effector proteins in plant-pathogenic fungi

“…Unlike fungal effectors, oomycete effectors frequently have conserved motifs including RxLR-dEER [ 16 ] and LXLFLAK (Crinklers/CRN) [ 17 ]. Traditional biochemical and structural analyses are the gold standard for the functional characterization of effector candidates [ 18 ] but are unsuitable for high-throughput analyses. Moreover, existing high-throughput experimental methods, such as proteomics and genome-wide association studies, routinely return numerous genes or proteins that may be associated with the phenotype of interest, necessitating some additional information to prioritize future experimental validation.…”

Chromosome-level genome assembly and manually-curated proteome of model necrotroph Parastagonospora nodorum Sn15 reveals a genome-wide trove of candidate effector homologs, and redundancy of virulence-related functions within an accessory chromosome

“…SNOG_03746 encodes a knottin-like protein. Knottins are cytotoxins that are best represented by snake and arachnid venoms, with the first fungal report of a knottin in the poplar rust Melampsora larici-populina [ 64 ]. SNOG_30910 encodes a homolog to phospholipase A2 - which cleaves sn-2 acyl bond between 2 phospholipids that releases arachidonic acid and lysophosphatidic acid - and is also a common domain in spider, insect and snake venoms that disrupt cell membranes [ 65 ].…”

Proteinaceous effector discovery and characterization in filamentous plant pathogens

“…One, MLP124266, is the first fungal protein to present a knottin‐like structure (Postic et al ., 2017) whilst the other, MLP1124499, shares structural similarity with members of the Nuclear Transport Factor‐2 (NTF2) superfamily. In both cases these candidate effectors show no sequence homology with structurally similar proteins and are the first examples of effectors with these structures (de Guillen et al ., 2019).…”