Deciphering Changes in Plant Physiological Response to Whitefly Feeding Using Microarray Technology

Gene array technology was applied to tomato as a model system to examine plant physiological response to silverleaf whitefly (SLW), Bemisia argentifolii (a.k.a. Bemisia tabaci biotype B) feeding. Our objective was to study gene expression in tomato plants subjected to a moderate infestation of whitefly. Plants were destructively harvested 25 days after infestation and samples of old and young leaves, stems, roots, flowers and fruit from tomato with and without whitefly were processed for nutritional analysis and RNA extraction for microarray analysis. RNA was labeled and hybridized to the gene array membrane to determine which genes SLW feeding influences. At 25 days after infestation, no discernable differences could be detected between plants with and without whitefly with the exception of uninfested plants possessing more flower buds. Whitefly pressure at harvest was moderate: 0.25 eggs per 50 mm, 0.04 nymphs per 50 mm and 0.52 adult whiteflies per leaflet. Plant nutritional profiles for plants with and without whitefly were not significantly different and were adequate for tomato production. RNA profiling results indicated 277 genes were up or down regulated in response to whitefly feeding and based on sequence similarity analysis we determined that selected genes were likely to be involved in developmental regulation, stress response, wound response, and ethylene production. INTRODUCTION Increased whitefly-related crop damage has been attributed to the establishment of a new whitefly biotype (B) which was subsequently described as a new species, the silverleaf whitefly (SLW) Bemisia argentifolii Bellows & Perring (Perring et al., 1993; Bellows et al., 1994). The SLW or B biotype has an expanded host range, increased intercrop mobility, and enhanced ability to develop insecticide resistance. In addition to damaging crops by sucking plant phloem and excreting honeydew which damages fruit and cotton by harboring sooty mold, the spread of geminiviruses in beans and tomatos (Blair et al., 1995; Polston and Anderson, 1997) was also associated with the introduction of this new B. tabaci biotype. An additional problem associated with the SLW was the appearance of a previously undescribed tomato irregular ripening disorder and a squash silverleaf disorder that appeared with the introduction of this exotic biotype in Florida (Schuster et al., 1990, 1991). Using tomato as a model system, our objective was to study gene expression in plants subjected to a moderate infestation of whitefly to decipher changes in tomato physiological response to whitefly feeding using microarray technology. MATERIALS AND METHODS Insect Source and Environmental Conditions Adult male and female B. tabaci biotype B (a.k.a B. argentifolii) were obtained from a laboratory colony maintained by the United States Horticultural Research Laboratory, Fort Pierce, Florida on dwarf cherry tomato (Lycopersicon esculentum 347 Proc. 1st IS on Tomato Diseases Eds. M.T. Momol, P. Ji and J.B. Jones Acta Hort 695, ISHS 2005 ‘Florida Lanai’) by continuous serial transfer since 1999. Dwarf cherry tomatos were infested with 5 adult whiteflies per leaf in Environmental Growth Chambers (EGC) (Environmental Growth Chambers, Chagrin Falls, Ohio) at the 5 true leaf stage (25 per plant). Control plants were kept in separate EGC with the same exact location in each chamber and environmental settings. Fluctuating temperature of 20°C night; 28°C day and fluctuating humidity from 80% at night; 40% in the day and 12:12 L:D photoperiod, light intensity satisfied all environmental settings during the experiment. All plants received the same water and fertilizer amounts at the same time. Soil and nutritional requirements were satisfied by standard ProMix Bx potting medium and fertilized weekly with Peter’s Professional General Purpose FLA Special 20-10-20. Insect, Plant Parameter Measurements and Nutritional Analysis Immature whitefly counts (eggs and nymphs) were taken randomly from leaves of each treatment with a # 5 cork borer between the leaf midveins. Each leaf disc measured 50 mm and was stored in ethanol for latter processing under the microscope. Adult whiteflies were counted on 100 leaflet turns per treatment. Individual tomato leaflets were randomly selected from leaves prior to turning to insure no bias in counting. All whitefly parameters were sampled 4, 11, 18, and 25 days after whitefly infestation. Tomato plants were sampled for height, width, and number of leaves, flower buds, flowers and fruit per plant on 0, 7, 16 and 25 days after whitefly infestation. Flowers were tagged at dehiscence so that fruit of the same age could be compared. Four plants from uninfested and infested plants were destructively sampled for nutritional analysis 25 days after whitefly infestation. Essential nutrients P, K, Ca, Mg, Fe, Mn, Zn, Cu, B, and Mo were analyzed by ICP (ThermoElemental, Madison, Wisconsin) following foliar tissue digestion by microwave. Total N analysis was performed utilizing a Thermo Quest NC 2100 Analyzer (CE Elantech Inc., Lakewood, New Jersey). RNA Profiling Using Microarray Technology Plants were destructively sampled for molecular analysis 25 days after whitefly infestation and samples included new leaf growth (top 3 leaves), old leaf growth (bottom 3 leaves), roots, stems, flowers, and fruit of each tomato plant. Three plants were pooled together as one replication to insure adequate plant material for molecular analysis and to increase sample homogeneity. Each treatment was replicated 3 times (9 plants per treatment). Live plants were destructively harvested and flash frozen in liquid nitrogen then immediately ground using a mortar and pestle, adding liquid nitrogen as needed to insure samples were processed in a frozen state. Samples were stored in a -80°C freezer until ready for processing. For all samples, equal amounts of tissue from the three experimental replications was combined, mixed thoroughly and sampled for total RNA extraction. Total RNA was extracted from old and young leaf tissue, root, stems, flower and fruit samples from uninfested and infested treatments. All total RNA samples were used to hybridize to previously purchased tomato microarrays from the Center for Gene Expression Profiling (CGEP). CGEP is a not-for-profit service facility and supported by the Boyce Thompson Institute at Cornell University and an NSF Major Research Infrastructure grant. Microarrays were processed at the University of Miami Medical Center Microarray Facility, Miami, Florida. Statistical Analysis Plant, insect, and nutritional data were analyzed by analysis of variance (ANOVA) to determine the effect of treatment. Calculations were performed with the general linear model (GLM) procedure of SAS (SAS Institute, Inc., Cary, NC.) with mean separation (P ≥ 0.05) by Duncan’s multiple range test or Ryan-Einot-Gabriel-Welsch multiple-range test (REGWQ) where appropriate. RESULTS AND DISCUSSION At 25 days after infestation, no discernable differences could be detected between