Candidate grapevine rust effectors suppress AvrB-triggered plant cell death in laboratory assay

A transcriptomic, microscopy and functional study of the grapevine rust fungus Neophysopella tropicalis identified 303 candidate effectors expressed during urediniospore germination, of which six suppressed AvrB-triggered hypersensitive cell death when delivered heterologously into Nicotiana benthamiana leaves. The study used a single fungal isolate, Niagara Rosada grape leaves and a bacterial delivery system, so its findings define an early candidate repertoire rather than directly establishing the mechanisms of virulence in grapevine. Among the 303 proteins predicted from the fungal transcriptome, 142 putative sequence families were detected, and expression profiling associated individual candidates with germination, host penetration or early colonization.

The researchers multiplied isolate Aglr64 of N. tropicalis on leaves of the American grape cultivar Vitis labrusca cv. Niagara Rosada, collected urediniospores and incubated them on polystyrene plates. After 16 hours at 22 degrees Celsius, 88% of the spores had developed long germ tubes.

Germinated spores were collected for RNA sequencing because this approach allowed pathogen RNA to be isolated without the large amount of plant material that would otherwise contaminate the sample. Paired-end sequencing generated more than 90 million trimmed reads. Assembly with Trinity produced 64,333 transcripts, including 28,968 longer than 1,000 base pairs, while BUSCO analysis indicated that 89.6% of the assembly contained sequences corresponding to conserved fungal single-copy genes.

The transcriptome also yielded full-length coding sequences for two beta-tubulin genes and a glyceraldehyde-3-phosphate dehydrogenase gene. One beta-tubulin sequence, NtBetaTub1, shared 100% identity with a previously reported N. tropicalis gene, whereas NtBetaTub2 was described as a possible second isoform not previously reported in the species. Two glyceraldehyde-3-phosphate dehydrogenase contigs differed by a single nonsynonymous substitution and were interpreted as allelic variants of a single-copy gene.

The researchers used these sequences, together with a cytochrome c oxidase subunit III reference, to normalize expression measurements. The complete beta-tubulin sequences may also support monitoring of mutations associated with benzimidazole fungicide resistance, although the present study did not test resistance. Of the 64,333 assembled transcripts, 6,081, or 9.45%, encoded predicted secreted proteins without a transmembrane domain.

Applying EffectorP with a probability threshold greater than 0.8 reduced this collection to 303 candidate effectors. After predicted signal peptides were removed, the mature protein sequences were clustered at a 40% global amino-acid identity threshold. The candidates formed 142 putative families: 65 contained a single member, while the other 77 contained between two and seven.

This distribution shows that the predicted repertoire was dominated by distinct and relatively small sequence groups rather than a few large expanded families. Seven of the 15 most highly expressed candidates had no detectable homologs in the National Center for Biotechnology Information non-redundant protein database. Those 15 candidates, designated NtEC-01 through NtEC-15, represented 13 sequence families and provided a broad sample of the predicted repertoire.

Four were homologous only to proteins from rust fungi, three had similarities restricted to Basidiomycota, and NtEC-10 showed sequence similarity across Basidiomycota and Ascomycota. Reverse-transcription PCR produced the expected mature coding-region amplicons for 14 candidates. NtEC-06 instead produced a product of approximately 380 base pairs rather than the predicted 480 base pairs, corresponding to an overall validation rate of 93.33% for the computational predictions.

These results establish the presence and relative abundance of candidate transcripts during spore germination; they do not by themselves demonstrate secretion, delivery or activity of each protein during infection. Reverse-transcription quantitative PCR separated the candidates into six expression patterns. NtEC-08 accumulated transcripts in both dormant and germinated spores, whereas NtEC-07 peaked in germinated spores and at 24 hours post-inoculation.

NtEC-02, NtEC-03, NtEC-10, NtEC-04, NtEC-11 and NtEC-13 reached their highest transcript levels in germinated spores. NtEC-01 and NtEC-14 peaked at 24 hours post-inoculation; NtEC-05 and NtEC-12 peaked at both 24 and 48 hours; and NtEC-09 and NtEC-15 peaked at 48 hours. Seven candidates were therefore associated chiefly with penetration and early biotrophic colonization.

Chitin-targeted fluorescence microscopy provided a developmental framework for interpreting those measurements. At 24 hours post-inoculation, N. tropicalis had formed elongated germ tubes ending in differentiated appressoria, with hyphae entering host tissue through stomatal openings. Similar structures remained visible at 48 hours, when intracellular infection hyphae indicated a transition from penetration to early colonization.

At 72 hours, infection hyphae were proliferating within the leaf, and by five days they had formed broad, coalescing intercellular networks. Closed and partly open uredia containing urediniospores appeared at seven days, while mature open pustules were present at nine days. Under these experimental conditions, penetration occurred at 24–48 hours, colonization was observed from 72 hours to five days, and reproduction began at seven to nine days.

Functional testing used the type III secretion system of Pseudomonas fluorescens EtHAn to deliver nine selected NtEC proteins, without their native signal peptides, into N. benthamiana leaves. Each candidate was co-infiltrated with AvrB, a bacterial protein that triggers a hypersensitive response. Suppression was scored when a candidate reduced or prevented tissue collapse in at least 40% of co-infiltrated leaves.

Across three independent experiments with ten biological replicates each, NtEC-05, NtEC-09 and NtEC-10 produced suppression frequencies of 53% to 66%, categorized as moderately high activity. NtEC-11, NtEC-12 and NtEC-13 produced frequencies of 40% to 46%, categorized as moderate activity. NtEC-03, NtEC-14 and NtEC-15 remained below 30% and were considered inactive in this assay.

AlphaFold 3 models offered preliminary structural information about the six candidates that reduced cell death. Predictions for NtEC-10 and NtEC-12 had high confidence, with predicted TM-scores above 0.5, while models for NtEC-05, NtEC-09, NtEC-11 and NtEC-13 had scores below 0.5 and were classified as low confidence. NtEC-10 showed sequence and structural similarity to a predicted glycan-binding protein from Phakopsora pachyrhizi.

That comparison may guide later biochemical work, but it does not establish glycan binding or any other biochemical activity. The study also did not determine whether the NtEC proteins act on RIN4, another immune regulator or through another route. Their measured ability to suppress an AvrB-triggered response only shows activity in the tested heterologous delivery assay.

Several scope limitations constrain interpretation of the results. The research used one N. tropicalis isolate and one susceptible grape cultivar, while the functional assays took place in N. benthamiana rather than grapevine. The experiment did not show that any candidate is required for virulence, that the proteins are secreted during natural infection or that they directly suppress immunity in grapevine.

Most expression peaks occurred during germination or early infection, and none of the selected candidates showed preferential expression at the later reproductive stages. Even so, the study assembled the first broad transcriptomic inventory of predicted N. tropicalis effectors, mapped candidate expression against an observed infection timeline and experimentally identified six proteins with cell-death-suppressing activity. These resources provide defined targets for subsequent work on secretion, host interactions and resistance-relevant effector recognition, without yet establishing a clinical, agricultural or disease-control outcome.

Subject of Research: Candidate effector proteins and stage-specific expression in the grapevine rust fungus Neophysopella tropicalis

Article Title: Grapevine rust fungus Neophysopella tropicalis deploys protein effectors with plant immunity-suppressing activity during sequential stages of pathogenesis

Article References: Maia, T., Ferro, J. V., Husein, G., Bombardelli, R. G. H., & Monteiro-Vitorello, C. B. (2026). Grapevine rust fungus Neophysopella tropicalis deploys protein effectors with plant immunity-suppressing activity during sequential stages of pathogenesis. Plant Cell Reports, 45(11), Article 337. https://doi.org/10.1007/s00299-026-03980-w

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03980-w

Keywords: Neophysopella tropicalis, grapevine leaf rust, fungal effectors, plant immunity, urediniospores, transcriptomics, host penetration, hypersensitive response, AlphaFold 3, biotrophic fungi, Grapevine, rust