Botrytis cinerea is a major plant pathogen infecting more than 1400 plant species. During invasion, the fungus rapidly kills host cells, which is believed to be supported by induction of programmed ...plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established which allowed to perform serial marker-free mutagenesis to generate multiple deletion mutants lacking up to 12 CDIPs. Whole genome sequencing of a 6x and 12x deletion mutant revealed a low number of off-target mutations which were unrelated to Cas9-mediated cleavage. Secretome analyses confirmed the loss of secreted proteins encoded by the deleted genes. Infection tests with the mutants revealed a successive decrease in virulence with increasing numbers of mutated genes, and varying effects of the knockouts on different host plants. Comparative analysis of mutants confirmed significant roles of two polygalacturonases (PG1, PG2) and the phytotoxic metabolites botrydial and botcinins for infection, but revealed no or only weak effects of deletion of the other CDIPs. Nicotiana benthamiana plants with mutated or silenced coreceptors of pattern recognition receptors, SOBIR1 and BAK1, showed similar susceptibility as control plants to infection by B. cinerea wild type and a 12x deletion mutant. These results raise doubts about a major role of manipulation of these plant defence regulators for B. cinerea infection. Despite the loss of most of the known phytotoxic compounds, the on planta secretomes of the multiple mutants retained substantial phytotoxic activity, proving that further, as yet unknown CDIPs contribute to necrosis and virulence. Our study has addressed for the first time systematically the functional redundancy of fungal virulence factors, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins to achieve necrotrophic infection of a wide variety of host plants.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The introduction of site-specific fungicides almost 50 years ago has revolutionized chemical plant protection, providing highly efficient, low toxicity compounds for control of fungal diseases. ...However, it was soon discovered that plant pathogenic fungi can adapt to fungicide treatments by mutations leading to resistance and loss of fungicide efficacy. The grey mould fungus
Botrytis cinerea
, a major cause of pre- and post-harvest losses in fruit and vegetable production, is notorious as a ‘high risk’ organism for rapid resistance development. In this review, the mechanisms and the history of fungicide resistance in
Botrytis
are outlined. The introduction of new fungicide classes for grey mould control was always followed by the appearance of resistance in field populations. In addition to target site resistance,
B. cinerea
has also developed a resistance mechanism based on drug efflux transport. Excessive spraying programmes have resulted in the selection of multiresistant strains in several countries, in particular in strawberry fields. The rapid erosion of fungicide activity against these strains represents a major challenge for the future of fungicides against
Botrytis
. To maintain adequate protection of intensive cultures against grey mould, strict implementation of resistance management measures are required as well as alternative strategies with non-chemical products.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The radio science experiment RSI on-board Rosetta determined the mass of the nucleus of comet 67P/Churyumov–Gerasimenko at the start of the prime mission from 2014 August to November (GM = 666.2 ± ...0.2 m3 s−2 or 9982 ± 3 × 1012 kg) and shortly before the end of the mission from 2016 July to September (GM = 665.5 ± 0.1 m3 s−2 or 9971.5 ± 1.5 × 1012 kg). The mass-loss is ΔM = 10.5 ± 3.4·109 kg, about 0.1 per cent of the nucleus mass. Almost 50 per cent of the mass-loss occurred during the 32 d before and 62 d after perihelion. The nucleus mass combined with the new very precise nucleus volume of 18.56 ± 0.02 km3 yields a bulk density of 537.8 ± 0.6 kg m−3. This low bulk density suggests that the nucleus is highly porous. The porosity is constrained by the observed bulk density, the density of ices, mostly water ice, and the density of compacted nucleus dust material. For a range of compacted dust material density from 2000 to 3500 kg m−3, the porosity varies between 65–79 per cent when the dust-to-ice mass ratio Fnucleus for the nucleus body lies in the range 3 ≤ Fnucleus ≤ 7. The nucleus is thus a highly porous very dusty body with very little ice. The total mass-loss ΔM puts hard constraints on the models of interpretation of the observations from other instruments on Rosetta. The loss from gas, based on ROSINA and MIRO observations, suggests that Fcoma, the dust-to-gas mass ratio of cometary matter in the coma beyond the Hill sphere of the nucleus is of the order of Fcoma ≤ 0.5 and therefore at least six times, and perhaps as much as 14 times, smaller than Fnucleus. It seems that the lost gas mass was overestimated by the instruments. The lost gas mass shall be 5.25 × 109 kg for Fcoma = 1. For any other gas mass values > 5.25 × 109 kg Fcoma is < 1. Most of the lifted dust mass will not leave the cometary Hill sphere but falls back to the nucleus. For 3 ≤ Fnucleus ≤ 7 and Fcoma = 0.5, the fallback mass is between 1.8·ΔM and 4.3·ΔM.
CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on ...the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The mitogen-activated protein kinase (MAPK) cassette of the cell wall integrity (CWI) pathway is primarily responsible for orchestrating changes of cell wall. However, functions of this cassette in ...other cellular processes are not well understood. Here, we found that the Botrytis cinerea mutant of MAPK kinase (BcMkk1) displays more serious defects in mycelial growth, conidiation, responses to cell wall and oxidative stresses, but possesses less reduced virulence than the mutants of its upstream (BcBck1) and downstream (BcBmp3) kinases. Interestingly, BcMkk1, but not BcBck1 and BcBmp3, negatively regulates production of oxalic acid (OA) and activity of extracellular hydrolases (EHs) that are proposed to be virulence factors of B. cinerea. Moreover, we obtained evidence that BcMkk1 negatively controls OA production via impeding phosphorylation of the Per-Arnt-Sim (PAS) kinase BcRim15 by the Ser/Thr kinase BcSch9. In addition, the fungal Pro40 homolog BcPro40 was found to interact simultaneously with three MAPKs, implying that BcPro40 is a scaffold protein of the CWI pathway in B. cinerea. Taken together, results of this study reveal that BcMkk1 negatively modulates virulence via suppressing OA biosynthesis in B. cinerea, which provides novel insight into conserved and species-specific functions of the MAPK kinase in fungi.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Retrotransposons are genetic elements inducing mutations in all domains of life. Despite their detrimental effect, retrotransposons can become temporarily active during epigenetic reprogramming and ...cellular stress response, which may accelerate host genome evolution. In fungal pathogens, a positive role has been attributed to retrotransposons when shaping genome architecture and expression of genes encoding pathogenicity factors; thus, retrotransposons are known to influence pathogenicity.
We uncover a hitherto unknown role of fungal retrotransposons as being pathogenicity factors, themselves. The aggressive fungal plant pathogen, Botrytis cinerea, is known to deliver some long-terminal repeat (LTR) deriving regulatory trans-species small RNAs (BcsRNAs) into plant cells to suppress host gene expression for infection. We find that naturally occurring, less aggressive B. cinerea strains possess considerably lower copy numbers of LTR retrotransposons and had lost retrotransposon BcsRNA production. Using a transgenic proof-of-concept approach, we reconstitute retrotransposon expression in a BcsRNA-lacking B. cinerea strain, which results in enhanced aggressiveness in a retrotransposon and BcsRNA expression-dependent manner. Moreover, retrotransposon expression in B. cinerea leads to suppression of plant defence-related genes during infection.
We propose that retrotransposons are pathogenicity factors that manipulate host plant gene expression by encoding trans-species BcsRNAs. Taken together, the novelty that retrotransposons are pathogenicity factors will have a broad impact on studies of host-microbe interactions and pathology.
Summary
Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near‐finished genome sequence for B. cinerea strain B05.10 is ...reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk ...of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
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The Botrytis cinere a VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their ...molecular basis.
Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed.
VELVET mutants showed reduced release of citric acid, the major acid secreted by the wildtype, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with 15N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation.
The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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