RESUMO
Phytophthora infestans is the cause of late blight, a devastating and re-emerging disease of potato. Significant advances have been made in understanding the biology of P. infestans, and in the development of molecular tools to study this oomycete. Nevertheless, little is known about the molecular bases of the establishment or development of disease in this hemibiotrophic pathogen. Suppression subtractive hybridization (SSH) was used to generate cDNA enriched for sequences upregulated during potato infection. To identify pathogen-derived cDNAs, and eliminate host sequences from further study, SSH cDNA was hybridized to a P. infestans bacterial artificial chromosome library. A new gene family was identified called Pinci1, comprising more than 400 members arranged in clusters of up to nine copies throughout the P. infestans draft genome sequence. Real-time RT-PCR was used to quantify the expression of five classes of transcript within the family, relative to the constitutively expressed PiactA gene, and it revealed them to be significantly upregulated from 12 to 33 h post-inoculation, a period defining the biotrophic phase of infection. Computational analysis of sequences suggested that transcripts were non-protein coding, and this was confirmed by transient expression of FLAG-tagged ORFs in P. infestans.
Assuntos
DNA de Algas/genética , Regulação da Expressão Gênica , Phytophthora/genética , RNA não Traduzido/genética , Solanum tuberosum/microbiologia , Sequência de Bases , DNA de Algas/química , Dosagem de Genes , Perfilação da Expressão Gênica , Biblioteca Gênica , Genoma , Dados de Sequência Molecular , Phytophthora/citologia , RNA de Algas/análise , RNA de Algas/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The oomycete Phytophthora infestans causes late blight, the potato disease that precipitated the Irish famines in 1846 and 1847. It represents a reemerging threat to potato production and is one of >70 species that are arguably the most devastating pathogens of dicotyledonous plants. Nevertheless, little is known about the molecular bases of pathogenicity in these algae-like organisms or of avirulence molecules that are perceived by host defenses. Disease resistance alleles, products of which recognize corresponding avirulence molecules in the pathogen, have been introgressed into the cultivated potato from a wild species, Solanum demissum, and R1 and R3a have been identified. We used association genetics to identify Avr3a and show that it encodes a protein that is recognized in the host cytoplasm, where it triggers R3a-dependent cell death. Avr3a resides in a region of the P. infestans genome that is colinear with the locus containing avirulence gene ATR1(NdWsB) in Hyaloperonospora parasitica, an oomycete pathogen of Arabidopsis. Remarkably, distances between conserved genes in these avirulence loci were often similar, despite intervening genomic variation. We suggest that Avr3a has undergone gene duplication and that an allele evading recognition by R3a arose under positive selection.
Assuntos
Proteínas de Algas/genética , Apoptose/genética , Phytophthora/genética , Phytophthora/patogenicidade , Solanum tuberosum/microbiologia , Agrobacterium tumefaciens , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biolística , Cromossomos Artificiais Bacterianos , Citoplasma/metabolismo , Primers do DNA , Duplicação Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Potexvirus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Solanum tuberosum/genética , Sintenia/genética , VirulênciaRESUMO
Phytophthora infestans, the causal agent of potato and tomato late blight, produces several different cell types prior to and during the early stages of potato infection. All of these cell types can be easily generated and studied in the absence of the host plant and so form the basis for developmental stage-specific gene discovery. We have used amplified fragment length polymorphism (AFLP)-based mRNA fingerprinting (cDNA-AFLP) to identify 64 transcripts that appeared to be up-regulated in germinating cysts but not in vegetative mycelium. These transcripts included representatives of most major classes of heat shock proteins: hsp60, hsp70, hsp90, and hsp100. Real-time RT-PCR was used to quantify the expression of 18 transcripts originating from germinating cysts, relative to the constitutively expressed actB gene, in vegetative mycelium, germinating cysts, and at three time-points post-inoculation of potato cultivar Bintje (15, 48, and 72h). All of the transcripts were up-regulated in germinating cysts, and 12, including hsp70, hsp80-2, and hsp90, were found also to be up-regulated in planta. This is the first report of the application of real-time RT-PCR to the relative quantification of plant pathogen gene expression during the early stages of infection.