1887

Abstract

is the cause of late blight, a devastating and re-emerging disease of potato. Significant advances have been made in understanding the biology of , 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 bacterial artificial chromosome library. A new gene family was identified called 1, comprising more than 400 members arranged in clusters of up to nine copies throughout the 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 A 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 .

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2007-03-01
2019-10-19
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Analysis of /potato interaction SSH using the following probes in Southern hybridizations: cDNA, cDNA; gDNA, genomic DNA; Binf , SSH cDNA. All cDNAs used as hybridization probes were digested with I and amplified with primer 1 from the Clontech PCR-Select cDNA subtraction kit following the ligation of both adaptors from the same kit. Lanes: M, 100 bp ladder (New England Biolabs); 1, uninfected potato cv Bintje (B0); 2, Bintje, 15 h post-infection (hpi) with P. infestans (B15); 3, Bintje 72 hpi with (B72); 4, Bintje, 9 hpi with (Ber); 5, Binf .

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UPGMA cluster analysis of BAC clones hybridizing to the Binf probe. DNA from all BAC clones was digested with dIII. Clusters were verified in all three instances by PCR from BAC end sequences; positive PCR amplification is marked by vertical lines, with the BAC end sequence marked on the right.

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Fingerprints of 10 overlapping BAC clones (from left to right): 56N16, 66N16, 68C21, 29G21, 56A9, 65A8, 12L6, 30M17, 54K5 and 26M13 digested with dIII, I and HI (A) and hybridized with a probe to 1-1 (B). Restriction fragment sizes in kb are marked on the right.

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Alignment of 1-1 to 1-25 (accession numbers EF091715 to EF091740) cDNA sequences amplified by RT-PCR.

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Alignment of deduced protein sequences from ORF1 from 19 members of the 1 family.

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Schematic diagram indicating the locations of blastn matches to 1, blastn matches to unigene sequences from Randall (2005) and predicted RXLR-containing ORFs on each of the 135 draft genome supercontigs containing matches to 1. Supercontigs are sorted in descending order of total length and are represented by three tracks, the topmost indicating locations of RXLR sequences (orange), the middle track representing matches to 1 (full-length matches in green, 5' domain matches in red and 3' domain matches in blue), and the lower track indicating matches to unigenes (purple). Scale bars are marked at every tenth supercontig. The clustering of matches to 1 sequences, and their co-location with unigenes are readily visible. The distribution of predicted RXLR sequences is also seen not to be associated with the distribution of 1 matches.

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Oligonucleotide primers used in sequencing and PCR.

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