Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

Genetic Diversity and Fungicide Sensitivity of Cytospora plurivora on Peach.

Cytospora plurivora D.P. Lawr., L.A. Holland & Trouillas has been associated with recent premature peach tree decline in South Carolina, but very little is known about the pathogen or chemical control options. Ninety-three C. plurivora isolates were collected in 2016 and 2017 from 1-year-old peach wood and symptomatic scaffold limbs, respectively, from orchards in six towns in South Carolina. Six unique genotypes were identified based on substantial ITS1-5.8S-ITS2 sequence variability and classified G1 to G6. Three of the genotypes (G2, G3, and G6) were isolated in high frequency in multiple locations of both years. In addition to the genotypic variation, multiple phenotypes were observed between and within genotype groups. Species identity was determined using additional gene loci: ACT, TUB, and EF, and isolates were found to belong to C. plurivora for all genotype groups. All tested genotypes were sensitive to thiophanate-methyl (FRAC 1) but exhibited slightly lower sensitivity to propiconazole and difenoconazole (both FRAC 3). Boscalid, fluopyram (both FRAC 7s), azoxystrobin, and pyraclostrobin (both FRAC 11s) were ineffective in vitro at inhibiting mycelial growth of C. plurivora genotypes. Field inoculation of peach and nectarine trees revealed that all genotypes developed twig cankers with differences in virulence. G1 was most virulent, and G6 was least virulent. This study provides a link between the C. plurivora genetic variability and virulence and provides fungicide sensitivity information that could be used to improve disease management practices.

Evaluation of the Intelligent Sprayer System in Peach Production.

Air-blast sprayers are routinely used to apply pesticides in commercial peach orchards, with growers using both conventional air-blast and ultrasonic sensor-driven models. Even with advanced spray technologies, there are still concerns with the amount of chemicals used and lost to drift. Our study evaluated a laser sensor-based variable-rate sprayer system in three experimental peach orchards for pest and brown rot disease control, spray volume output, spray coverage, and spray drift. A single 378-liter air-blast sprayer was used for both the conventional air-blast and the Intelligent Sprayer (iSprayer) treatments. Treatments were started at the phenological stage of bloom and continued through final swell. The iSprayer treatment was as effective in controlling pests and brown rot disease as the conventional air-blast treatment. Compared with the conventional air-blast treatment, the iSprayer treatment reduced the spray volume (liters/hectare) in cultivar PF23 by 71% at bloom, 62% at pit hardening, and 55% at final swell. For Juneprince, the spray volume reduction was 50% at bloom, 40% at pit hardening, and 13% at final swell. Spray drift was significantly (P < 0.05) reduced only at bloom in the iSprayer treatment. Spray coverage was increased by 50.13 and 26.67% in the iSprayer treatment at bloom and pit hardening, respectively, but not at final swell. Our results show that the iSprayer maintained pest and disease control efficacy in peach orchards while reducing spray volume and drift compared with the conventional air-blast treatment.

Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement.

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

Novel gene-sequence markers for isolate tracking within Monilinia fructicola lesions.

BACKGROUND: Monilinia fructicola is a diverse pathogen of pome and stone fruits that causes severe economic losses each year. However, little is known about inoculum flow within or between orchards and pathogen establishment in an orchard, because few methods exist for detecting diversity or tracking isolates over time. SSR loci are an effective option, but may be confounded by a high degree of mutability and potential sensitivity to abiotic stress. RESULTS: Through transcriptome analysis, we identified novel markers mrr1, DHFR and MfCYP01 and validated stability of these markers under fungicide stress in natural infection sites. Nucleotide variation within mrr1, DHFR and MfCYP01 sequences differentiated isolates at all spatial scales: within the same infection site, between trees and between two farms. Sequenced regions were also effective for matching isolates collected from blossoms at the beginning of the season to progeny in cankers obtained at the end of the season. CONCLUSIONS: Collectively, results show that mrr1, DHFR and MfCYP01 are able to accurately differentiate M. fructicola isolates at the population level, can be used to track isolates over time, and are more stable than SSRs under external stresses. Either by themselves or combined with SSR markers, these gene-encoding regions are a much-needed tool for better understanding M. fructicola population dynamics. © 2017 Society of Chemical Industry.