Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae

Abstract

Filamentous plant pathogens deliver effector proteins into host cells to suppress host defence responses and manipulate metabolic processes to support colonization. Understanding the evolution and molecular function of these effectors provides knowledge about pathogenesis and can suggest novel strategies to reduce damage caused by pathogens. However, effector proteins are highly variable, share weak sequence similarity and, although they can be grouped according to their structure, only a few structurally conserved effector families have been functionally characterized to date. Here, we demonstrate that Zinc-finger fold (ZiF) secreted proteins form a functionally diverse effector family in the blast fungus Magnaporthe oryzae. This family relies on the Zinc-finger motif for protein stability and is ubiquitously present in blast fungus lineages infecting 13 different host species, forming different effector tribes. Homologs of the canonical ZiF effector, AVR-Pii, from rice infecting isolates are present in multiple M. oryzae lineages. Wheat infecting strains of the fungus also possess an AVR-Pii like allele that binds host Exo70 proteins and activates the immune receptor Pii. Furthermore, ZiF tribes may vary in the proteins they bind to, indicating functional diversification and an intricate effector/host interactome. Altogether, we uncovered a new effector family with a common protein fold that has functionally diversified in lineages of M. oryzae. This work expands our understanding of the diversity of M. oryzae effectors, the molecular basis of plant pathogenesis and may ultimately facilitate the development of new sources for pathogen resistance.

Fig 1. Mutations in AVR-Pii Zinc-finger motif abrogate binding to Exo70 host target.

(A) Yeast two-hybrid assay of AVR-Pii and AVR-Pii^CCH with OsExo70F2 or OsExo70F3 host targets. For each combination, 5μl of yeast were spotted and incubated in double dropout plate for yeast growth control (left) and quadruple dropout media supplemented with X-α-gal and 3AT (right). Growth, and development of blue coloration, in the selection plate are both indicative of protein:protein interaction. OsExo70 proteins were fused to the GAL4 DNA binding domain and AVR-Pii to the GAL4 activator domain. Empty vectors were used as negative control in each combination. (B) Co-immunoprecipitation of AVR-Pii and AVR-Pii^CCH with OsExo70F2. N-terminally GFP-tagged AVR-Pii effectors were transiently co-expressed with N-terminally 3xFLAG-tagged OsExo70F2 or 3xFLAG-mCherry in N. benthamiana. Immunoprecipitates (IPs) were obtained with anti-FLAG magnetic beads and total protein extracts were probed with appropriate antisera.

Fig 2. AVR-Pii Zinc-finger Fold (ZiF) defines an effector family in M. oryzae.

(A) Workflow of HMM-based search for Zinc-finger fold effectors. Schematic representation of the ZiF motif search pipeline and results. (B) Superimposition of the ZIF fold of AVR-Pii (light brown) and representative members of all ZIF effector tribes of M. oryzae. (C) Alphafold2 models of amino acid residues that form the binding interface and the zinc finger motif. Amino acid variation within tribes is shown in parentheses. The experimental model of AVR-Pii (PDB: 7PP2) is added as a reference.

Fig 3. Zif effectors are conserved across different host-specific lineages of M. oryzae.

Presence/absence analysis of ZiF effector tribes across host-specific lineages of M. oryzae. An ASTRAL multispecies coalescence tree (reprinted of a figure panel from Bentham et al. [21]) is shown on the left. On the right, we indicate the presence of ZiF effectors for each tribe in every blast isolate. ZiF tribes without presence in the genome of a blast isolate is represented as white. The presence of a pseudogenized effector (no start codon and/or truncation of the ZiF fold) are represented in grey. When more than one member of the same ZiF effector tribe is present, it is represented in different shades of blue.

Fig 4. Wheat blast AVR-Pii homolog ZiF_VIIIc binds to OsExo70F3 and is recognized by Pii resistance in rice.

(A) Y2H assay of wild-type AVR-Pii and ZiF_VIIIc and their corresponding mutants at the host target binding interface (Phe65Glu and Phe66Glu, respectively) to host target OsExo70F3. Left, control plate for yeast growth. Right, quadruple-dropout media supplemented with X-α-gal and aureobasidine A (Au A). Growth and development of blue coloration in the right panel indicates protein-protein interactions. OsExo70F3 was fused to the GAL4 DNA binding domain while effectors were fused to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Rice leaf blade spot inoculation of transgenic M. oryzae Sasa2 isolates expressing AVR-Pii or ZiF_VIIIc from wheat blast isolate BTJP 4–1 in rice cultivars Moukoto (Pii-) and Hitomebore (Pii+). The cultivars Moukoto and Hitomebore are denoted by M and H, respectively. For each experiment, a representative image from replicates with independent M. oryzae transformants are shown. Wild-type rice blast isolate Sasa2 and wheat blast Br32 and BTJP 4–1 are included as control. Full images for the three experimental replicates are presented in S8 Fig. (C) Spray inoculation of transgenic M. oryzae isolates in 3-weeks-old rice cultivar Moukoto and Hitomebore. Each experiment was performed four times (two of the leaves are shown for the Sasa2, Br32 and BTJP 4–1 isolates). All leaf images are shown in S9 Fig.

Fig 5. ZiF effectors do not share the same host target.

(A) Y2H binding assay of ZiF effectors from rice blast isolates to host target OsExo70F3. For each tribe, the most prevalent allele in rice blast lineages was fused to the GAL4 activator domain and co-expressed in yeast cells with OsExo70F3 fused to GAL4 DNA binding domain. AVR-Pii Phe65Glu was used as negative control as previously reported [43]. (B) Y2H binding assay of ZiF effectors of wheat blast isolates to host target OsExo70F3. The most prevalent effector allele in each ZiF tribe of M. oryzae isolates infecting wheat was fused to the GAL4 activator domain and co-expressed in yeast cells with OsExo70F3 fused to GAL4 DNA binding domain. Rice blast AVR-Pii and AVR-Pii Phe65Glu were used as positive and negative controls, respectively. For both assays, a control plate for yeast growth is presented on the left and a plate with quadruple-dropout media supplemented with X-α-gal and aureobasidine A (Au A) is presented on the right. Growth and development of blue coloration in the right panel indicates protein-protein interactions. Each experiment was repeated a minimum of three times, with similar results.