Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

Genetic Composition of Phytophthora infestans in Canada Reveals Migration and Increased Diversity.

A dramatic increase in the incidence of late blight and changes within populations of Phytophthora infestans have been observed in various regions of Canada. In this study, the occurrence of several new genotypes of the pathogen was documented with associated phenotypes that dominated pathogen populations. Genotype US-23, previously detected only among isolates from the United States, dominated in the western Canadian provinces of British Columbia, Alberta (AB), Saskatchewan, and Manitoba (MB). Although isolates of US-23 infect both potato and tomato, these isolates were the only genotype recovered from commercial garden centers in Canada. Isolates of genotype US-8, previously dominant throughout Canada, represented the only genotype detected from the eastern Canadian provinces of New Brunswick and Prince Edward Island. Isolates of other genotypes detected in Canada included US-11 in AB, US-24 in MB, and US-22 in Ontario (ON). An additional genotype was detected in ON which appears to be a derivative of US-22 that may have arisen through sexual reproduction. However, evidence of clonal reproduction dominated among the isolates collected, and opportunities for sexual reproduction were probably limited because of a surprising geographic separation of the A1 and A2 mating types in Canada. Sensitivity of the US-22, US-23, and US-24 isolates to the fungicide metalaxyl, movement of potato seed and transplants, and weather conditions may have contributed to reduced opportunities for contact between the mating types in fields in Canada. All P. infestans isolates were readily distinguished from other related oomycetes with RG57 restriction fragment length polymorphism analysis. Long-distance movement in seed tubers and garden center transplants may have contributed to the rapid spread of the P. infestans genotypes across Canada. Tracking pathogen movement and population composition should improve the ability to predict the genotypes expected each year in different regions of Canada.

In-Furrow Applications of Metalaxyl and Phosphite for Control of Pink Rot (Phytophthora erythroseptica) of Potato in New Brunswick, Canada.

The efficacy of metalaxyl-m (Ridomil Gold 480EC) and phosphite (Phostrol) applied at planting in-furrow against pink rot (Phytophthora erythroseptica) of potato (Solanum tuberosum) 'Shepody' and 'Russet Burbank' was evaluated in field trials conducted in 2005 and 2006 in Florenceville, New Brunswick, Canada. Inoculum made from a metalaxyl-m-sensitive isolate of P. erythroseptica from New Brunswick was applied either in-furrow as a vermiculite slurry at planting or as a zoospore drench in soils adjacent to potato plants in late August. After harvest, the number and weight of tubers showing pink rot symptoms were assessed and expressed as percentages of the total tuber number and total weight of tubers. Metalaxyl-m applied in-furrow was significantly more effective against pink rot than phosphite. The mean percentage of diseased tubers as a percentage of total tuber weight was 1.5% (2005) and 1.2% (2006) for metalaxyl-m-treated plots and 9.6% (2005) and 2.8% (2006) for phosphite-treated plots, a percentage similar to that obtained in inoculated control plots with no fungicide treatment. The mean percentage of diseased tubers expressed as a percentage of the total number of tubers was 1.7% (2005) and 1.3% (2006) for metalaxyl-m-treated plots and 10.1% (2005) and 3.1% (2006) for phosphite-treated plots. Disease incidence was significantly higher using the late-season inoculation technique (respective means in 2005 and 2006 were 9.9 and 3.8% diseased tubers, by weight, and 10.6 and 3.9%, by number) than with the in-furrow inoculation method (respective means in 2005 and 2006 were 3.3 and 0.7% by weight, and 3.7 and 1.3%, by number). The potato cv. Shepody was significantly more susceptible to pink rot (9.9 and 3.3% diseased tubers, by weight, in 2005 and 2006, respectively, and 10.6 and 3.9%, by number) than Russet Burbank (respective means in 2005 and 2006 were 3.4,% and 1.2%, by weight, and 3.7,% and 1.2%, by number). Our findings indicate that metalaxyl applied in-furrow at planting is a viable option for control of pink rot caused by metalaxyl-sensitive strains of P. erythroseptica, whereas phosphite was ineffective.

Proteomics analysis suggests broad functional changes in potato leaves triggered by phosphites and a complex indirect mode of action against Phytophthora infestans.

Phosphite (salts of phosphorous acid; Phi)-based fungicides are increasingly used in controlling oomycete pathogens, such as the late blight agent Phytophthora infestans. In plants, low amounts of Phi induce pathogen resistance through an indirect mode of action. We used iTRAQ-based quantitative proteomics to investigate the effects of phosphite on potato plants before and after infection with P. infestans. Ninety-three (62 up-regulated and 31 down-regulated) differentially regulated proteins, from a total of 1172 reproducibly identified proteins, were identified in the leaf proteome of Phi-treated potato plants. Four days post-inoculation with P. infestans, 16 of the 31 down-regulated proteins remained down-regulated and 42 of the 62 up-regulated proteins remained up-regulated, including 90% of the defense proteins. This group includes pathogenesis-related, stress-responsive, and detoxification-related proteins. Callose deposition and ultrastructural analyses of leaf tissues after infection were used to complement the proteomics approach. This study represents the first comprehensive proteomics analysis of the indirect mode of action of Phi, demonstrating broad effects on plant defense and plant metabolism. The proteomics data and the microscopy study suggest that Phi triggers a hypersensitive response that is responsible for induced resistance of potato leaves against P. infestans. BIOLOGICAL SIGNIFICANCE: Phosphie triggers complex functional changes in potato leaves that are responsible for the induced resistance against Phytophthora infestans. This article is part of a Special Issue entitled: Translational Plant Proteomics.