No abstract

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.

SUMMARYEfficient nutrient acquisition is critical to the fitness of plant pathogens. To address how the late blight agent Phytophthora infestans adapts to nutrients offered by its hosts, genes in glycolytic, gluconeogenic and amino acid pathways were mined from its genome and their expression in different plant tissues and artificial media was measured. Evidence for conventional glycolytic and gluconeogenic processes was obtained, although several steps involved pyrophosphate-linked transformations which are uncommon in eukaryotes. In media manipulation studies, nearly all genes in the pathways were subject to strong transcriptional control. However in rye-sucrose media, tomato leaflets, potato tubers and, at both early and late stages of infection, most glycolytic genes were expressed similarly, which indicated that each plant tissue presented a nutrient-rich environment. Biochemical analyses also demonstrated that sporulation occurred from host material in which sugars were abundant, with fructose and glucose increasing at the expense of sucrose late in the disease cycle. The expression of only a few genes changed late in infection, with the most notable example being lower invertase levels in the sucrose-reduced leaves. Interestingly, most gluconeogenic genes were up-regulated in tubers compared with other tissues. Rather than reflecting a starvation response, this probably reveals the role of such enzymes in converting carbon skeletons from the abundant free amino acids of tubers into citric acid cycle and glycolysis intermediates, as genes involved in amino acid catabolism were also more highly expressed in tubers. The corresponding enzymes also displayed higher activities in defined media when amino acids were abundant, as in tubers.m pp_570 843..856

Transcriptional changes during asexual sporangia formation by the late blight pathogen Phytophthora infestans were identified using microarrays representing 15,646 genes and RNA from sporulation time-courses, purified spores, and sporulation-defective strains. Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato. During sporulation, about 12% of genes were up-regulated compared to vegetative hyphae and 5% were down-regulated. The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport. Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia. For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia. Data from this study are a resource for understanding sporogenesis, which is critical to the pathogenic success of P. infestans and other oomycetes.