Analysis of the Molecular Dialogue Between Gray Mold (Botrytis cinerea) and Grapevine (Vitis vinifera) Reveals a Clear Shift in Defense Mechanisms During Berry Ripening.

Mature grapevine berries at the harvesting stage (MB) are very susceptible to the gray mold fungus Botrytis cinerea, while veraison berries (VB) are not. We conducted simultaneous microscopic and transcriptomic analyses of the pathogen and the host to investigate the infection process developed by B. cinerea on MB versus VB, and the plant defense mechanisms deployed to stop the fungus spreading. On the pathogen side, our genome-wide transcriptomic data revealed that B. cinerea genes upregulated during infection of MB are enriched in functional categories related to necrotrophy, such as degradation of the plant cell wall, proteolysis, membrane transport, reactive oxygen species (ROS) generation, and detoxification. Quantitative-polymerase chain reaction on a set of representative genes related to virulence and microscopic observations further demonstrated that the infection is also initiated on VB but is stopped at the penetration stage. On the plant side, genome-wide transcriptomic analysis and metabolic data revealed a defense pathway switch during berry ripening. In response to B. cinerea inoculation, VB activated a burst of ROS, the salicylate-dependent defense pathway, the synthesis of the resveratrol phytoalexin, and cell-wall strengthening. On the contrary, in infected MB, the jasmonate-dependent pathway was activated, which did not stop the fungal necrotrophic process.

The homeobox BcHOX8 gene in Botrytis cinerea regulates vegetative growth and morphology.

Filamentous growth and the capacity at producing conidia are two critical aspects of most fungal life cycles, including that of many plant or animal pathogens. Here, we report on the identification of a homeobox transcription factor encoding gene that plays a role in these two particular aspects of the development of the phytopathogenic fungus Botrytis cinerea. Deletion of the BcHOX8 gene in both the B. cinerea B05-10 and T4 strains causes similar phenotypes, among which a curved, arabesque-like, hyphal growth on hydrophobic surfaces; the mutants were hence named Arabesque. Expression of the BcHOX8 gene is higher in conidia and infection cushions than in developing appressorium or mycelium. In the Arabesque mutants, colony growth rate is reduced and abnormal infection cushions are produced. Asexual reproduction is also affected with abnormal conidiophore being formed, strongly reduced conidia production and dramatic changes in conidial morphology. Finally, the mutation affects the fungus ability to efficiently colonize different host plants. Analysis of the B. cinerea genome shows that BcHOX8 is one member of a nine putative homeobox genes family. Available gene expression data suggest that these genes are functional and sequence comparisons indicate that two of them would be specific to B. cinerea and its close relative Sclerotinia sclerotiorum.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination.

The genetic diversity of three temperate fruit tree phytoplasmas 'Candidatus Phytoplasma prunorum', 'Ca. P. mali' and 'Ca. P. pyri' has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of 'Ca P. prunorum' the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of 'Ca. P. prunorum', the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese 'Ca. P. pyri' isolates showed that they shared some alleles with 'Ca. P. prunorum', supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Striking diversity of vmp1, a variable gene encoding a putative membrane protein of the stolbur phytoplasma.

Studies of phytoplasma-insect vector interactions and epidemiological surveys of plant yellows associated with the stolbur phytoplasma (StolP) require the identification of relevant candidate genes and typing markers. A recent StolP genome survey identified a partial coding sequence, SR01H10, having no homologue in the "Candidatus Phytoplasma asteris" genome but sharing low similarity with a variable surface protein of animal mycoplasmas. The complete coding sequence and its genetic environment have been fully characterized by chromosome walking. The vmp1 gene encodes a protein of 557 amino acids predicted to possess a putative signal peptide and a potential C-terminal transmembrane domain. The mature 57.8-kDa VMP1 protein is likely to be anchored in the phytoplasma membrane with a large N-terminal hydrophilic part exposed to the phytoplasma cell surface. Southern blotting experiments detected multiple sequences homologous to vmp1 in the genomes of nine StolP isolates. vmp1 is variable in size, and eight different vmp1 RsaI restriction fragment length polymorphism types could be distinguished among 12 StolP isolates. Comparison of vmp1 sequences revealed that insertions in largest forms of the gene encode an additional copy of a repeated domain of 81 amino acids, while variations in 11-bp repeats led to gene disruption in two StolP isolates. vmp1 appeared to be much more variable than three housekeeping genes involved in protein translation, maturation, and secretion and may therefore be involved in phytoplasma-host interactions.

A chromosome map of the Flavescence doree phytoplasma.

The Flavescence dorée phytoplasma (FD-P), a non-cultivable, plant-pathogenic bacterium of the class Mollicutes, is the causal agent of a quarantine disease affecting vineyards of southern Europe, mainly in southern France and northern Italy. To investigate FD-P diversity and phytoplasma genetic determinants governing the FD-P life cycle, a genome project has been initiated. A physical map of the chromosome of FD-P strain FD92, purified from infected broad beans, was constructed by performing restriction digests of the chromosome and resolving the fragments by PFGE. Single and double digestions of the chromosome with the enzymes SalI, BssHII, MluI and EagI were performed and used to map 13 restriction sites on the FD-P chromosome. The size of the chromosome was calculated to be 671 kbp. Southern blot analyses using cloned phytoplasma probes were carried out to assist in the arrangement of contiguous restriction fragments and to map eight genetic loci, including the two rRNA operons, the tuf, uvrB-degV and secY-map (FD9) genes, the FD2 marker and two orphan sequences (FDDH29 and FDSH05) isolated through subtractive suppression hybridization.

Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class III gene (Bcchs3a).

Botrytis cinerea is an important phytopathogenic fungus requiring new methods of control. Chitin biosynthesis, which involves seven classes of chitin synthases, could be an attractive target. A fragment encoding one of the class III enzymes was used to disrupt the corresponding Bcchs3a gene in the B. cinerea genome. The resulting mutant exhibited a 39% reduction in its chitin content and an 89% reduction in its in vitro chitin synthase activity, compared with the wild-type strain. Bcchs3a mutant was not affected in its growth in liquid medium, neither in its production of sclerotia, micro- and macroconidia. In contrast, the mutant Bcchs3a was severely impaired in its growth on solid medium. Counterbalancing this defect in radial growth, Bcchs3a mutant presented a large increase in hyphal ramification, resulting in an enhanced aerial growth. Observations by different techniques of microscopy revealed a thick extracellular matrix around the hyphal tips. Moreover, Bcchs3a mutant had a largely reduced virulence on Vitis vinifera and Arabidopsis thaliana leaves.

Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity.

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity.

Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species. Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized. Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine (Vitis vinifera) suspension cells, and the mutant-plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1. Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.