Unravelling Signalling Processes in the Potato R Gene Resistance Response to Phytophthora infestans

vokKJB. Yksikön huom.: KJ

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for large-scale studies of oomycete genome organisation and gene content

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals

Large-scale gene discovery in the oomycete Phytophthora infestans reveals likely components of phytopathogenicity shared with true fungi

o overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans¿potato and ¿tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coverage of the P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pathogenicity, such as class III pectate lyases, despite the considerable evolutionary distance between oomycetes and fungi. Thirty-seven genes encoding components of flagella also were identified. Several genes not anticipated to occur in oomycetes were detected, including chitin synthases, phosphagen kinases, and a bacterial-type FtsZ cell-division protein. The sequence data described are available in a searchable public database

Differential gene expression, induced by salicylic acid and Fusarium oxysporum f. sp. lycopersici infection, in tomato Expressão diferencial de genes induzida por ácido salicílico e por Fusarium oxysporum f. sp. lycopersici, em tomateiro

The objective of this work was to determine the transcript profile of tomato plants (Lycopersicon esculentum Mill.), during Fusarium oxysporum f. sp. lycopersici infection and after foliar application of salicylic acid. The suppression subtractive hybridization (SSH) technique was used to generate a cDNA library enriched for transcripts differentially expressed. A total of 307 clones was identified in two subtractive libraries, which allowed the isolation of several defense-related genes that play roles in different mechanisms of plant resistance to phytopathogens. Genes with unknown roles were also isolated from the two libraries, which indicates the possibility of identifying new genes not yet reported in studies of stress/defense response. The SSH technique is effective for identification of resistance genes activated by salicylic acid and F. oxysporum f. sp. lycopersici infection. Not only the application of this technique enables a cost effective isolation of differentially expressed sequences, but also it allows the identification of novel sequences in tomato from a relative small number of sequences.<br>O objetivo deste trabalho foi determinar o perfil de transcritos em plantas de tomate (Lycopersicon esculentum Mill.), durante a infecção com Fusarium oxysporum f. sp. lycopersici e após a aplicação foliar de ácido salicílico. A técnica de hibridização subtrativa por supressão (SSH) foi utilizada para gerar uma biblioteca de cDNA enriquecida por transcritos diferencialmente expressos. Foram identificados 307 clones, em duas bibliotecas subtrativas, que permitiram o isolamento de diversos genes de defesa com função em diferentes processos relacionados à resistência vegetal contra patógenos. Também foram isolados, nas duas bibliotecas, genes com função desconhecida, o que indica a possibilidade de identificação de novos genes que ainda não tenham sido relatados em estudos anteriores de resposta a estresses e defesa, em plantas. A técnica SSH é eficiente em identificar genes de resistência, ativados pelo ácido salicílico e pela infecção com Fusarium oxysporum f. sp. lycopersici. A aplicação dessa técnica não apenas possibilita isolar seqüências diferencialmente expressas, a baixo custo, como também permite a identificação de novas seqüências, em tomate, a partir de um número relativamente pequeno de seqüências