1a. Objectives (from AD-416):
1. Characterize the kinetics of host ROS production and changes in the expression of genes related to ROS production or scavenging in fruit tissue as a response to yeast antagonist cells and exogenous ROS. Focus will be on NADPH oxidase, SOD, and peroxidase. 2. Characterize the effect of ROS on host genes associated with MAPK signaling cascade leading to host defense reactions. Focus will be on salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) orthologues in apple and citrus fruit tissue. 3. Determine the implications of elevated ROS production (induced by antagonist yeasts or other factors) at the infection sites (surface wounds) on the ability of pathogenic fungi to infect host tissue. 4. Examine the effects of ROS on the tolerance of yeast antagonists to stress conditions (dehydration, nutrient starvation, osmotic stress) and on their ability to proliferate, colonize, and form biofilms in or on wounded and intact fruit tissue, respectively. In this regard, we will examine the activity and gene expression of three enzymes known to be involved in oxidative and stress tolerance in yeasts: catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX), and also, characterize the overall proteomic response of yeast antagonists to exogenous ROS.
1b. Approach (from AD-416):
Kinetics and mechanism of ROS production and scavenging in fruit tissue as affected by yeast antagonist cells (Candida oleophila and Metschnikowia fructicola), fruit type (apple and citrus), and maturity/ripening stage will be studied using assays for superoxide anions and hydrogen peroxide, laser confocal microscopy, apple and citrus microarrays, and Real-Time qPCR analysis of the expression of oxidative stress related genes. Expression of key genes in the MAPK signaling cascade (orthologues of SIPK and WIPK in apple and citrus) will be investigated by means of Real-Time qPCR. The implication of ROS in wound sites on infection and development of the pathogens (Penicillium digitatum and Penicillium expansum) will be assessed using various elicitors and inhibitors of ROS. Production of ROS by yeast cells, and the expression and activity of ROS-related genes and proteins, respectively, will be determined using Real-Time qPCR techniques and spectrophotometric assays. DIGE technology and MALDI-TOF analysis will be utilized to investigate changes in the yeast proteome as affected by ROS and other stressors.
3. Progress Report:
The work conducted at ARO laboratory during the second year was focused on characterization the molecular and biochemical responses of citrus fruit tissue to the application of the yeast M. fructicola. For this purpose, we used RNA samples collected from grapefruit peel tissue during the previous fruit season (2009/2010) and also additional samples collected during the 2010/2011 season. An Affymetrix Citrus GeneChip microarray was used to characterize gene expression. The biochemical work included the tests aimed at determination of the kinetics of ROS (H2O2, superoxide and hydroxyl radical) in fruit tissue using specific stains and confocal laser microscopy as well as colorimetric reactions. Expression of genes of interest (oxidative, signaling and defense–related genes) were studied using qRT-PCR. The work conducted at the AFRS laboratory focused on evaluating the effect of high temperature and oxidative stress on the cell viability and biocontrol efficacy of the yeast M. fructicola. Studies were conducted to determine the effect of heat shock on accumulation of ROS in yeast cells as well as expression of genes related to stress tolerance [e.g. trehalose-6-phosphate synthase (TPS1)]. Effect of both heat shock and oxidative stress on biocontrol activity against P. expansum and P. digitatum on apples and citrus fruit, respectively, was also characterized.