Generation and analysis of expressed sequence tags from Botrytis cinerea

Abstract

Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.

Transcriptome Profiling of Botrytis cinerea Conidial Germination Reveals Upregulation of Infection-Related Genes during the Prepenetration Stage

“…For hybridization, Nimblegene 4-plex arrays, containing 4 ϫ 72,000 arrays per slide, were used. Three 60-mer oligonucleotides per gene were immobilized for a total number of 20,885 predicted genes of the two sequenced B. cinerea strains B05.10 and T4, with 3,189 T4-specific genes and 1,776 B05.10-specific genes (designated BC1G_XXXX) (38). Genes annotated according to strain T4 (http://urgi .versailles.inra.fr/gb2/gbrowse/BOTRYTIS_T4_pub/) are referred to as in the abbreviated form PXXXXXX, instead of BofuT4_PXXXXXX.1, throughout this article.…”

Bchex virulence gene of Botrytis cinerea: characterization and functional analysis

“…The hex gene from B. cinerea was identified and isolated from a cDNA genomic library of this fungus (Silva et al 2006). The predicted protein HEX-1 from B. cinerea shares 71 and 76% identity with the HEX protein from N. crassa and M. grisea, respectively (Asiegbu et al 2004;Tenney et al 2000).…”

“…From a B. cinerea cDNA library described previously (Silva et al 2006), we identified a sequence highly homologous to the hex-1 gene from N. crassa (AF001033). Gene-specific primers were designed and used to amplify the Bchex gene from genomic DNA of B. cinerea strain A1.…”

“…Isolation, cloning and sequencing of Bchex-coding region from a B. cinerea cDNA library Specific primers H 1 and H 4 , which flank the Bchex-coding region, were designed from contig 609 obtained from the cDNA library (Silva et al 2006) ( Table 1). The PCR mixture contained one unit of Taq polymerase, 10 lM of each primer, 1.5 mM MgCl 2 ; 12.5 mM dNTPs, approximately 20-40 ng of B. cinerea genomic DNA and H 2 O.…”

“…In previous work, we described a preliminary analysis of an EST library from B. cinerea growing in axenic cultures. From this analysis, we identified the hex gene (Bchex) as being highly expressed during filamentous growth (Silva et al 2006). This gene, which encodes the principal protein of the Woronin body, is involved in hyphal growth.…”