Genotyping-by-Sequencing to Predict Resistance to Lima Bean Downy Mildew in a Diversity Panel.

Lima bean is affected by severe downy mildew epidemics caused by the oomycete Phytophthora phaseoli. There are six documented races of P. phaseoli (A to F). Race F is currently predominant in the mid-Atlantic region, creating the need for resistant lima bean cultivars with desirable agronomic characteristics. In order to develop markers for detecting race F resistance, bulked segregant analysis (BSA) using genotyping-by-sequencing (GBS) was used on a biparental F2 population comprised of 216 lima bean progeny segregating for a dominant race F resistance phenotype. Data were analyzed using a custom bioinformatic analysis pipeline (redrep). Kompetitive allele-specific polymerase chain reaction assays were developed using 12 GBS markers associated with the race F resistance phenotype. Using these assays, the F2 population was used to map the race F resistance locus. Seven markers were in linkage and significantly associated with race F resistance that mapped between two markers located approximately 4.88 centimorgan (cM) apart. These assays were successfully used to genotype a newly acquired lima bean diversity panel consisting of 256 landraces, cultivars, and wild germplasm, and a haplotype consisting of two of the seven linked markers was demonstrated to accurately predict race F resistance. This confirmed the ability of our customized methods to accurately predict phenotypes in diverse lines of lima bean.

Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea.

Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.

Salicylic acid and NIM1/NPR1-independent gene induction by incompatible Peronospora parasitica in arabidopsis.

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants.

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.

Investigating the efficacy of current quality assurance performance tests in diagnostic ultrasound.

The efficacy of four current quality assurance performance tests in diagnostic ultrasound was investigated. This was done by comparing the ability of the performance tests to detect fluctuations in gray scale, depth of penetration, axial resolution and lateral resolution on a real-time scanner with the ability of sonologists and sonographers to detect the same fluctuations. The performance tests for gray scale, depth of penetration, and axial resolution were found to be efficacious. The lateral resolution test was judged nonefficacious . This suggested a need to revise the protocol for the lateral resolution test with regard to real-time scanners.