2012 Annual Report
To understand R3bv2 cold tolerance, we will use a transcriptomic approach to compare total pathogen gene expression during infection of plants at 20°C (cool, permissive for R3bv2) and 28°C (warm, permissive for both R3bv2 and bv3 strains). We will use two complementary microarray chips custom-designed to represent the complete genomes of R3bv2 strain UW551 and bv3 strain GMI1000. Using a 4-way experimental design, we will compare gene expression of tropical strain GMI1000 and cool-temperate strain UW551 during plant infection under cool and warm conditions. This comparative analysis will identify candidate cold tolerance genes that can be tested for function using site-directed mutagenesis followed by virulence and survival assays at the two temperatures. Microarray data will also reveal larger patterns of pathway expression or suites of genes that are likely to play roles in this complex trait; the contribution of such patterns to cool-temperate bacterial wilt disease will be tested using rational mutagenesis designed to abrogate the pathway or trait in question.
To determine the biological basis of R3bv2 latent infection, we will draw on the custom-designed UW551 microarray chips developed above, we will compare R3bv2 pathogen gene expression during latent and active (symptomatic) infection of host plants. Identifying conditions that predictably produce latently infected plants has been a challenge, but cooler temperatures and lower inoculum are promising. A comparative analysis of total gene expression during the two kinds of infection will test the hypothesis that the pathogen exists in a different physiological and defensive state during latent infection. The practical purpose of these experiments is to identify chemical or environmental triggers that would either prevent or reverse latent infections, allowing offshore growers to block or expose latently infected plants before they could be accidentally introduced to the U.S.
Excluding R3bv2 from introduction into the U.S. demands detection methods that are sensitive but also practical so they can be used at offshore production sites. Despite glowing claims, many recently-published detection methods require costly elaborate equipment or reagents, and are not reproducible in our hands. Using infected geranium plants, we directly compared the speed, sensitivity, cost, and technical difficulty of four published methods and a new one that uses nucleic acid-binding cards followed by R3bv2-specific PCR. The card method is technically simple, identifies bacteria to the R3bv2 subgroup (not just to species like immunostrips), is less expensive than immunostrips, and kills the pathogen so the cards can be safely transported at room temperature before the PCR reaction. With a detection limit of 103 to 104 pathogen cells, the card-PCR method is 10-100 times as sensitive as the immunostrips, good enough to detect latently infected plants.
We used comparative in planta transcriptomic analysis to identify R3bv2 traits that contribute to this strain’s ability to cause potato brown rot at cool temperatures. A lectin (a sugar-binding protein) is involved in pathogen attachment to plant roots at cool temperatures, suggesting a potential disease control strategy. R3bv2’s differential ability to defuse host defenses may also increase cool virulence.