ABSTRACT: RESEARCH SUMMARY Bacterial blight and bacterial leaf streak, two of the most serious diseases of rice, are caused by Xanthomonas oryzae pathovar (pv.) oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) respectively. Xoo is a vascular pathogen that invades the xylem, whereas Xoc is non-vascular and colonizes the mesophyll apoplast. These distinctive types of infection represent the two most common ways plant pathogenic bacteria produce disease. As closely related pathogens of a model plant host, Xoo and Xoc constitute a powerful comparative system. We hypothesize that tissue-specific susceptibility to pathogenic bacteria can be explained partially by differential host responses during infection. To establish the global structure of susceptible responses to infection by Xoo and Xoc, we used the Affymetrix Rice Genome Array to analyze the abundance of transcripts in rice plants over four days after inoculation with virulent strains. MATERIAL AND METHODS - Plant Material: Oryza sativa ssp. japonica cv. Nipponbare plants were grown in pots containing LC-1 soil mixture, and arranged in flats of 20 pots, in growth chambers under a 12-h, 28ºC light and a 12-h, 25ºC dark cycle. To allow inversion of the flats for inoculation (modified dip-inoculation method, Niño-Liu et al, 2005, Journal of Phytopathology) seeds were sown through 1-cm perforations in a fibreglass tray resting on top of the pots. Peters Professional Fertilizer and iron chelate micronutrient were applied with watering every two days at rates of 0.25 and 4.5 g/L, respectively, until the day before inoculation. - Bacterial Strains and preparation of inoculum: Xoo strain PXO99A and Xoc strain BLS256 were used. For each, a single colony from a glucose yeast extract (GYE) agar plate was transferred to 5 ml of GYE liquid medium and incubated for 24 h at 28ºC with constant shaking at 250 rpm. Subsequently, 2 ml of this culture was transferred to 300 ml of fresh GYE liquid medium and incubated as above for an additional 18 h. Cells were pelleted by centrifugation at 4000 rpm. for 10 min, washed twice and resuspended in sterile 10 mm MgCl2 to an OD600 of 0.05, yielding 7 liters of inoculum. Tween-20 was added to a final concentration of 0.5%. - Modified dip-inoculation method: Fourteen days after sowing, and 2 h after the beginning of the light period, seedlings were inverted into inoculum, one flat each for Xoo, Xoc and Mock inoculum consisting of sterile 10 mM MgCl2 with 0.5% Tween-20, and vaccuum infiltrated at 500mm Hg for two minutes, two consecutive times, in a custom-made chamber. Following inoculation, plants were returned to the growth chamber. During growth, plants were cycled weekly in the same order through three growth chambers. In this way all plants could be incubated in the same final chamber following inoculation. For each replicate, position of the flats in the growth chamber (from left to right, used also for the order of inoculation and harvest) was determined by a split-plot design. - Timepoints, tissue collection: Within flats, assignment of pots for the harvest of inoculated tissue at 2, 4, 8, 24 and 96 h was made using a separate randomized complete block design. Tissue was harvested from plants in four pots (approximately 2g fresh weight) for each timepoint per inoculum per replication. Harvested tissue was immediately frozen in liquid nitrogen and stored at -80ºC until processing. - RNA isolation, purification, labeling, hybridizations, scanning: Total RNA was initially isolated from frozen whole plant tissue using the hot TRIzol reagent protocol for barley as described by Caldo et al (Plant Cell, 2004). The RNA was purified using Qiagen RNeasy spin columns, and adjusted to a final concentration of 1 ug/uL. Probe synthesis, labeling, and hybridization were performed at the Iowa State University GeneChip Core facility. ACKNOWLEDGEMENTS This work was supported by a grant (0227357) from the Plant Genome Research Program of the National Science Foundation. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, David O Niño-Liu. The equivalent experiment is OS3 at PLEXdb.]
