Sequencing the Obligate Oomycete Plant Pathogen, Bremia lactucae

Bremia lactucae is an obligate biotrophic oomycete that causes downy mildew, the most serious disease of lettuce throughout the U.S. and worldwide.  In order to investigate the molecular basis of pathogenicity and host specificity, we are sequencing its genome and mining it for genes encoding candidate effectors.  We are assembling a draft genome sequence of B. lactucae isolate SF5 using Sanger- Illumina, and PacBio-generated sequences from several types of libraries.  Using Illumina reads, the nuclear genome has been assembled into 21,000 scaffolds, totaling 88 Mb with an N50 = 41.5 kb. The high level of heterozygosity has made assembly challenging and therefore we are assimilating other types of library and sequence.  The mitochondrial genome has also been assembled.  RNA-seq data is being generated to aid in annotating the genome.  We have also sequenced the gene-space of 11 diverse isolates to determine how representative the reference isolate is of the species.   Computational analysis has so far identified ~ 50 candidate effectors with RXLR motifs that are being functionally characterized for hypersensitive response (HR)-eliciting activity using Agrobacterium-mediated transient expression assays in a differential set of resistant lettuce cultivars and for HR suppression during co-expression with known HR elicitors.   So far, HR-eliciting activities have not been detected for 47 RXLR candidate effectors and one CRN-like protein.  Two NLPs were found to elicit cell death following over-expression in several lettuce genotypes as well as in Arabidopsis.  Several Y2H interactions have been detected between potential effectors from B. lactucae and candidate plant targets.