henhaobio
Sign in

bioRxivMolecular BiologyPreprint

Multi-knockout of 29 phytotoxic proteins and metabolites does not completely abolish virulence of the necrotrophic fungus Botrytis cinerea

Nassim SafariiD, Patrick Pattar, Marat Magomedov, Andrea Tobian Herreno, Shekhar Chule, Dolores Fernández‐OrtuñoiD, Luoyu wu, Frederik SommeriD, David ScheuringiD, Michael SchrodaiD, Remco StamiD, Matthias HahniD

PDFDOI
In-depth full-text digest available · Jump to digest ↓

Abstract

Botrytis cinerea is a necrotrophic plant pathogen with an extremely wide host range. During invasion, the fungus induces rapid host cell death and proliferates in the necrotic tissue. Host killing involves secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs), but their relative contributions are poorly understood. We have previously shown that the sequential knockout of up to 12 CDIPs leads to a substantial reduction of virulence of B. cinerea mutants. In this study, we identified additional CDIPs and generated an unprecedented series of multi-gene deletion mutants in a filamentous fungus, culminating in a 29x mutant carrying deletions of 27 CDIP-encoding genes and two genes required for the biosynthesis of the phytotoxins botrydial and botcinic acid. These multi-k.o. mutants were strongly reduced in virulence and almost unable to infect apple fruit tissue, but still induced slowly expanding necrosis on leaves, demonstrating that additional determinants of host killing remain to be identified. Overexpression of the highly phytotoxic Nep1 in a 22-fold CDIP mutant failed to increase its virulence. Reevaluation of several CDIPs previously described as virulence factors revealed for most of them only small or no significant contributions to pathogenesis. Generation of a mutant lacking all six predicted endo-polygalacturonases confirmed only for PG1 and PG2 a major role for cell wall degradation and infection. Our work demonstrates that necrotrophic pathogenesis in B. cinerea does not depend on a few primary virulence determinants, but rather on a highly redundant network of host damaging factors.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (27 Aug 2026) · CC BY 4.0

Deleting 27 CDIP-candidate genes (26 confirmed CDIPs) and two phytotoxin biosynthesis genes in Botrytis cinerea nearly abolishes apple infection but leaves slow leaf lesions, pointing to redundant, unidentified host-killing factors.

Single deletions of cell death inducing proteins (CDIPs) often gave no phenotype because of functional redundancy, and earlier sequential mutants lacking up to 12 CDIPs and phytotoxins still caused disease. This work extends the series to 29 deletions to test whether a few primary virulence determinants explain necrotrophy.

Key results

  1. ShownThe 29x mutant (27 CDIP genes plus the botrydial and botcinic acid biosynthesis genes deleted) caused almost no lesions on apple at 4 days but still produced slowly expanding lesions on bean and tomato leaves.

    Mutants from 17x upward almost abolished apple lesions at 4 days. On apple after 21 days, the 18x, 27x and 29x mutants formed similarly slow, hard lesions (n=6, no significant differences). The 18x mutant caused almost no lesions on Arabidopsis leaves. · Phenotypes of the newly generated 13x-29x…; Discussion; Supporting information

  2. ShownMost of the virulence loss traces to PG1, PG2 and the two phytotoxins. Deleting all six endo-polygalacturonases gave no loss beyond the pg1 pg2 double mutant.

    Virulence was only slightly reduced from 12xbb up to 16x, with a significant drop at 17x (PG2 deleted). Mutants from 17x to 29x were similar or slightly lower. Polygalacturonase activity fell similarly in pg1 pg2, 4xPG and 6xPG secretomes (n=3). · Phenotypes of the newly generated 13x-29x…; Elimination of all six endopolygalacturonases…; Discussion; Supporting information

  3. ShownThe 22x and 29x on planta secretomes retained about 25% of WT phytotoxicity; the residual activity of the 29x secretome is largely heat- and protease-sensitive.

    On planta secretomes from 22x and 29x retained about 25% of WT necrosis-inducing activity on N. benthamiana (n=10-14; the authors say precise comparison is not possible). For the 29x secretome, heating at 100°C for 2.5 min removed almost all activity; proteinase K reduced it significantly but not completely. · Analysis of the secretomes of B. cinerea WT and…; Figure legends

  4. ShownOverexpressing the strongly phytotoxic Nep1 in the 22x mutant did not raise secretome toxicity or virulence.

    With two strong promoters (H2B, His3), Nep1 was abundant by MS/MS in in vitro and on planta secretomes. Secretome toxicity on Vicia faba (n=6-7) and tomato lesion size (n=24-45 at 48 hpi) matched the 22x parent. · Overexpression of Nep1 in a 22x mutant does not…; Figure legends

  5. ShownRe-testing reported virulence factors with marker-free single mutants confirmed few effects. Only crh4 was clearly reduced; ssp2 on bean and IEB2 on tomato showed slight reductions.

    crh4 was significantly reduced on tomato and bean leaves (tomato n=22-60 per mutant, pooled WT n=113). CFEM1, Xyl1, Xyl2 and CELP1 mutants showed no apparent phenotype. The 21x mutant, with crh4 deleted, was not less virulent than 20x. · Generation of B. cinerea single marker-free CDIP…; Discussion; Figure legends

Methods, data and reproducibility

The authors extended a marker-free CRISPR-Cas9 knockout series from 12x to 29x, with 22 rounds of deletion, and screened homology- and AlphaFold-based CDIP candidates by infiltration and agroinfiltration. They compared mutants on several host tissues, by secretome proteomics and phytotoxicity, and by genome sequencing.

  • B. cinerea B05.10 WT and 13x to 29x multi-knockout mutants (bean, tomato, apple; the 18x also on maize, Arabidopsis and grape), plus PG mutants up to 6xPG (tomato, bean, apple) · Botrytis cinerea · n = Fig 2: bean n=20-73, tomato n=17-64, apple n=5-27 per genotype
  • Illumina genome sequencing of 18x, 23x, 28x and 29x mutants, about 60x coverage, aligned to the B05.10 reference · Botrytis cinerea · n = 4 mutant genomes
  • MS/MS label-free proteomics of on planta (tomato leaf) and in vitro secretomes of WT and mutants · Botrytis cinerea · n = WT on planta n=10-13; single 29x sample
  • Plant defence mutants tested for B. cinerea sensitivity (eds1 pad4 sag101a sag101b quadruple mutant; bak1, sobir1, eds1) · Nicotiana benthamiana; Arabidopsis thaliana · n = n=20-28
Methods & tools
CRISPR-Cas9 RNP editing, AlphaFold, FastQC v0.12.1, BWA-MEM v0.7.17, GATK HaplotypeCaller, snpEff, SPAdes, ImageJ, GraphPad Prism, MS/MS label-free proteomics, Agroinfiltration in N. benthamiana
Reagents & environment
FastQC v0.12.1, BWA-MEM v0.7.17, SPAdes with k-mer sizes 33 to 77; GATK and snpEff versions not stated.
Reproducibility gaps
No code repository, sequencing accession or proteomics deposit is stated. The transformation protocol is cited to a separate methods chapter (Zenodo record).
Limitations (4)
  • Multi-knockout phenotypes cannot be confirmed by complementation. Illumina sequencing cannot exclude large rearrangements or epigenetic changes. · stated by the authors
  • Weak effects of individual CDIPs are hard to detect when deleted in an already strongly attenuated multi-knockout background, so a combined analysis of single and multiple mutants is needed. · stated by the authors
  • WT and mutant secretomes were produced under different inoculation conditions, so the comparison of their phytotoxicity is only approximate. · stated by the authors
  • The proteomic confirmation that deleted CDIPs are absent from the 29x secretome rests on a single 29x on planta sample, and nep1 qRT-PCR came from one experiment. · noted in this summary

Read this if you study fungal necrotrophy or plant cell-death elicitors and want quantitative evidence on how redundant host-killing factors are in Botrytis cinerea.

Citations
1

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISLeisen '21Qin '25Mueller '23Zhu '21Anand '21Gupta '20
89% semantic matchMolecular Biology
Thomas Leisen, Janina Werner, Patrick Pattar+5 more·bioRxiv·Aug 21, 2021· 2 citations