In our experimental format, all three cultivars are susceptible to P. capsici. ‘New Yorker’ seeds were obtained from a commercial source . The homozygous ABA-deficient mutants sitiens and flacca were compared with their isogenic, wild-type background, ‘Rheinlands Ruhm,’ and seeds for these were obtained from the C. M. Rick Tomato Genetics Resource Center, University of California, Davis. ‘Rheinlands Ruhm,’ sitiens, and flacca plants were grown for seed production in the greenhouse. NahG transgenic plants were generated in the ‘New Yorker’ background, similar to the method used by Gaffney et al. . The nahG construct containing the transgene salicylate hydroxylase under control of the CaMV 35S promoter in the binary vector pCIB200 was a gift of Syngenta Crop Protection, Inc. SA deficiency of our transgenic line was confirmed previously . The acx1 and def1 mutants in the cv. ‘Castlemart’ background were a gift of Gregg Howe, Michigan State University. Seeds of ‘Castlemart’ were obtained from the C. M. Rick Tomato Genetics Resource Center. Experiments were conducted in a growth chamber .The salt stress regime selected for these experiments was based on prior studies of root stress predisposition . The impact of salinity stress differs from other osmotic dehydration stresses primarily in that salt-stressed plants are additionally exposed to abnormally high extracellular concentration of ions such as sodium and chloride . The inclusion of calcium helps to mitigate the confounding toxicity caused by sodium and emphasizes the osmotic facet of salinity stress,vertical indoor hydroponic system which is likely applicable to other dehydration stresses . Plants were subjected to salt stress by replacing the 0.5X Hoagland’s solution with 0.2 M NaCl and 0.02 M CaCl2 for 18 h. Plants were returned to 0.5X Hoagland’s solution, allowed to regain turgor and recover for 2 h, and then inoculated with zoospores of P. capsici .

To determine whether there was an effect on zoospore motility and chemotaxis, a microcapillary swim-in assay similar to that described by Morris and Ward was used with exudates collected from tomato roots. Following 18 h salt stress, tomato roots of uniform volume were rinsed in deionized H2O and transferred to tubes containing 2 ml of deionized water. Exudates were allowed to accumulate for 2 h, tomatoes were then removed, and the exudates were vortexed and immediately loaded into 1 µl microcapillary tubes . Exudate-loaded microcapillaries were placed into 15 cm petri dishes with one end submerged in a 500 µl droplet of 5 × 105 zoospores ml−1 . Microcapillaries were photographed under a dissecting microscope 15 min after being placed into the zoospore suspension. Zoospore attraction was determined as the proportion of the microcapillary’s inside diameter blocked by encysted zoospores and scored on a 0–5 rating scale . The P. capsici-GFP transformant was visualized 24 and 48 hours post inoculation in tomato roots using a Leica TCS SPE confocal system . Following infection and just prior to microscopy, roots were dipped into a 10 µg/ml solution of propidium iodide for 30 s and rinsed in sterile water before mounting on microscope slides . GFP was excited at 488 nm and emission was collected between 510 and 550 nm. PI was excited at 534 nm and emission was collected between 600 and 650 nm. Laser power was set to 50% with a gain of 800–900 for both the 488 nm and 534 nm channels. Final images were composites of five Z steps through root tissues approximately 40 µm in depth. To estimate the progression of P. capsici colonization in tomato seedlings by qPCR, nahG, def1, and acx1 plants and wild-type plants of their corresponding backgrounds were frozen in liquid N2 at 48 hpi, and stored at −80◦C until extraction and analysis. Samples for quantitation of P. capsici DNA were extracted and analyzed using the method described in Dileo et al. . For gene expression analyses, RNA was extracted from tomato seedlings using RNeasy Plant Mini kits according to the manufacturer’s instructions . Samples were obtained from roots pooled from fifive plants, with three samples for each treatment in each experiment.

Extracts weretreated with Dnase I to remove genomic DNA contaminants. Intact 25s and 18s ribosomal RNA bands were visualized by gel electrophoresis . cDNA stock solutions were prepared with the iScript cDNA synthesis kit . A complete list of target genes and primers can be found in Table 1. Gene expression was quantifified with a 7500 FAST Realtime PCR thermocycler , using SsoFAST EvaGreen Supermix with low Rox . Relative quantities were determined using the 11 CT method, normalizing against cyclophilin and uridylate kinase . Jasmonic acid was generated by base hydrolysis of methyl jasmonate [3-oxo-2-cyclopentaneacetic acid, methyl ester, 95% purity; Sigma-Aldrich] according to the procedure of Farmer et al. . The experimental treatment sequence was as follows. Roots of hydroponically grown tomato seedlings were immersed for 72 h in a solution of JA . Seedlings were removed from the JA solution and transferred to fresh 0.5X Hoaglands for 48 h, and then exposed to salt stress for 18 h as described above. After a 2 h recovery in 0.5X Hoaglands, the roots were inoculated with 1 × 104 zoospores/ml of P. capsici. Roots were then collected at 24 hpi for gene expression analyses as described above, with samples obtained from roots pooled from five plants and three samples analyzed for each treatment. JA at 25 µM was selected because higher concentrations were slightly phytotoxic in our experimental format. Disease assays in ‘New Yorker,’ ‘Rheinlands Ruhm,’ and ‘Castlemart’ backgrounds were performed three times, with five replicates for each treatment within each experiment. For ordinal data and for qPCR data that typically did not satisfy the analysis of variance criterion for normality, the Wilcoxon rank sums or Kruskal–Wallis tests were used for means comparisons. Gene expression time courses were performed twice. When data satisfied the criterion for normality, ANOVA and the Dunnett’s test or Student’s T-test were used for means comparisons. Analyses were performed with JMP Pro software . A brief episode of salt stress applied prior to inoculation of tomato seedlings with zoospores of P. capsici results in infections of greater severity and a classic predisposition phenotype . Previously, increased zoospore attraction was observed in salt-stressed chrysanthemum roots relative to non-stressed roots . To determine if salt-stress enhances the attraction of tomato roots to zoospores and whether ABA influences this, we used a quantitative chemotaxis choice assay to compare exudates from non-stressed and salt-stressed tomato roots. Exudates collected from ABAdeficient flacca and sitiens mutants and their background wild-type ‘Rheinlands Ruhm’ roots following salt stress were significantly more attractive to P. capsici zoospores than exudates collected from non-stressed roots. However, exudates from the ABA-defificient mutants, sitiens and flacca, were equally attractive as those collected from ‘Rheinlands Ruhm’ . ABA alone was not a chemo attractant in this assay, having a ZARS value of 0, the same as deionized water. We used confocal microscopy to further characterize root infections under our experimental regime to determine if salt stress of the host prior to inoculation causes P. capsici to change its infection and colonization strategy.

Examination of roots inoculated with a P. capsici-GFP strain 24 hpi revealed haustoria in host cells deep within the root tissue . Haustoria were observed in both salt-stressed and non-stressed roots, with the only apparent microscopic distinction between the treatments during the course of observation being the greater extent of colonization in salt-stressed roots. Propidium iodide , which stains nuclei in dead or dying cells, was used as a vital stain to assess root cell viability under the various treatments. Non-inoculated roots in the non-stressed and salt-stressed treatments were similar in appearance, with occasional PI-staining of nuclei . There was nonspecific staining of plant cell walls by PI in all treatments, which is common due to the exclusion of the dye from membranes of living cells that makes outlines of the cells visible. Inoculated, non-stressed roots were mostly intact with limited instances of PI staining of nuclei , while inoculated, salt-stressed roots contained numerous PI-stained nuclei . In both treatments, root tips and the bases of lateral roots were the most colonized regions. In a previous study, we found that ABA levels in tomato roots increase rapidly following exposure to salt stress and during the onset of predisposition, and then decline to near pre-stress levels . To determine if the expression of genes associated with ABA synthesis and response follows a similar course during stress onset and recovery, NCED and TAS14 were monitored by qPCR in tomato roots. NCED encodes the 9-cis-epoxycarotenoid-dioxygenase , a critical step in ABA biosynthesis and generally considered to be rate-limiting . TAS14 is a tomato dehydrin gene that is induced by salt stress and ABA, but not by cold or wounding,vertical farming tower for sale and serves as a salt stress-induced marker of ABA responses in tomato . NCED1 expression increased rapidly in tomato roots following salt exposure in a manner that generally corresponded with ABA measurements reported previously , and returned to pre-stress levels similar to ABA . Salt challenge of tomato roots induced TAS14 within 3 h after immersion of the roots in the salt solution, with maximum expression as much as ∼4,000-fold above the initial basal expression . NCED1 gene expression levels returned to basal levels 24 h following removal of the roots from the salt treatment , whereas TAS14 gene expression levels from the same plants returned to pre-stress values within 12 h of salt removal . The changes in TAS14 expression were limited to salt-stressed roots, as baseline expression in nonstressed roots was at or below the sensitivity of our analytical platform. P. capsici infection in either salt-stressed or non-stressed plants did not appear to influence NCED1and TAS14 expression. In tomato, P4 , a PR-1 ortholog, serves as a marker for induction of the SA pathway . P4 transcript accumulation was measured in non-stressed and salt-stressed ‘New Yorker’ tomato roots following inoculation with P. capsici. P4 was induced only in plants inoculated with P. capsici . Plants that had been salt-stressed prior to inoculation had significantly lower levels of P4 transcripts relative to non-stressed, inoculated plants . P4 expression remained suppressed even at 48 hpi in salt-stressed, inoculated plants. In tomato, proteinase inhibitor II is a wound and pathogen-inducible marker of JA responses . PI-2 showed a similar pattern of expression as P4 in our experimental regime and was induced only in P. capsici-inoculated plants . Prior salt stress resulted in significantly reduced PI-2 gene expression throughout the period of observation . Salt stress alone did not induce P4 or PI-2 expression. To determine if SA and JA influence the severity of disease susceptibility induced by salt-stress, tomato plants altered in SA levels and JA synthesis were evaluated in the predisposition assay.

NahG and WT tomatoes both displayed enhanced susceptibility following salt stress, but NahG plants had significantly higher basal susceptibility to P. capsici even without salt stress . Nonetheless, the proportional increase in P. capsici colonization in salt-treated plants relative to non-salted plants was similar in both the WT and NahG tomato genotypes. ‘Castlemart’ tomatoes, and the acx1 and def1 mutants within this genetic background, unlike other tomato genotypes we have used in predisposition studies, did not display a predisposition phenotype under our treatment regime . Colonization of these plants by P. capsici trended less in the salt-treated seedlings, and significantly less in salt-treated acx1 seedlings compared to non-salted plants . This was unexpected, rendering results with the def1 and acx1 mutants inconclusive relative to the issue of JA action in predisposition. Without suitable JA-deficient mutants available to this study, we then sought to determine whether exogenous JA could alter or override the salt stress inhibition of PI-2 gene expression using ‘New Yorker’ seedlings, which display a consistent and clear predisposition phenotype. Treatment of roots with exogenous JA strongly induced PI-2 transcripts, with salt treatment reducing transcript accumulation . The PI-2 expression pattern was similar in the inoculated seedlings pretreated with JA and/or salt. The tomato 13-LOX and 13-AOS genes encode key enzymes in JA biosynthesis . 13-LOX expression at the time of sampling was not significantly affected by any treatment . Although AOS transcript levels were relatively low in all treatment combinations, salt stress reduced AOS expression by more than half in both non-inoculated and inoculated seedling roots . This reduction was partially offset by JA pretreatment. P4 expression was not induced by JA, salt or their combination; however, inoculation with P. capsici following JA treatment resulted in a strong induction of P4 transcripts .