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 five plants, with three samples for each treatment in each experiment. Extracts were treated 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 quantified with a 7500 FAST Real time PCR thermocycler , using SsoFAST Eva Green 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.
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,cultivo de arandanos 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-deficient mutants, sitiens and flacca, were equally attractive as those collected from ‘Rheinlands Ruhm’ . ABA alone was not a chemoattractant 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, 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 non-stressed 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. 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 . Previous research in our laboratory demonstrated that tomato seedling roots and crowns became highly susceptible to P. capsici following a brief exposure of the roots to salt stress . These plants generally regained turgor during the course of the stress treatment,macetas de plastico but remained in a predisposed state in the absence of visible stress symptoms for up to 24 h following removal from the salt. The salt stress effect on disease appears to operate through an ABA dependent mechanism, as evidenced by the loss of predisposition in ABA-deficient mutants and partial complementation with exogenous ABA to restore the predisposition phenotype . Salinity stress also has been shown to make roots more attractive to Phytophthora zoospores . In the present study, chemoattraction of P. capsici zoospores to exudates from salt-stressed roots was significantly greater than to exudates from non-stressed roots. However, exudates from salt-stressed roots of wild-type tomato plants and ABA-deficient mutants were equally attractive . Thus, differences in root attraction to zoospores cannot explain the differences in disease severity between wild-type and ABA-deficient plants.
These results reinforce our view that the determinative effects of stress-induced ABA in predisposition occur during infection, invasion and colonization, rather than during pre-infection events related to root exudation, zoospore attraction and initial contact with the root . Our results also affirm an earlier study on salinity-induced susceptibility to Phytophthora root rot that pointed to a strong effect of the stress on host defenses .P. capsici is a hemibiotroph, establishing haustoria in host cells during the early stages of infection, and then necrotizing host tissue as the infection progresses . Confocal imaging revealed the presence of haustoria in infected tomato roots that appeared as simple protrusions into root cells , closely resembling those described in the literature for Phytophthora haustoria . After reviewing dozens of P. capsici infections in non-stressed and salt-stressed roots, we concluded that haustoria are present in both treatments. Therefore, it does not appear that P. capsici alters its fundamental infection strategy in salt-stressed tomato roots. The only clear distinction apparent between treatments was the increased rate of colonization, as reflected in greater abundance of hyphae in the salt-stressed roots relative to the controls. While the pathogen’s infection strategy does not appear to change, based on microscopic examination, it is possible that P. capsici alters its strategy in other ways, such as the timing or pattern of display of effectors. We attempted to measure expression of putative and known P. capsici effector genes believed to correspond to the switch from bio-trophy to necrotrophy . Pathogen RNA proved difficult to recover during early infection and later as plant tissues died, and so we were unable to detect alterations in effector expression as a function of treatment. Transcriptome analyses using deep sequencing as reported in a study of P. capsici on tomato leaves may prove to be better able to address this question . Endogenous ABA levels are tightly regulated in the plant by balancing biosynthesis, catabolism and conjugation . NCED1 expression in roots during the 18 h salt stress treatment generally corresponded with salt-induced ABA accumulation that we reported in our previous study . Similar findings in Phaseolus vulgaris showed stress-induced expression of NCED, with accumulation of NCED protein and ABA occurring within a 2 h window . While stimuli have been described that upregulate NCED1 gene expression, relatively little information is available regarding mechanisms for its down regulation. In drought-stressed Arabidopsis, ABA production and expression of NCED3 is correlated with the level of available carotenoid substrates . NCED1 expression in tomato roots may diminish as ABA levels decline or as external stresses are removed. Possible post-transcriptional and/or post-translational regulation of NCED1/NCED cannot be ruled out, as suggested for regulation of AAO , the terminal step in ABA synthesis . Following an episode of salt stress and inoculation with P. capsici, NCED1 transcript levels returned to pre-stress levels in tomato roots and remained at basal levels in all treatments throughout the 48 h infection time course . However, we saw no evidence for NCED1 induction or ABA accumulation during infection with P. capsici. This is in contrast to Arabidopsis infected by Pst, which induces AtNCED3 and ABA accumulation in leaves . Expression of TAS14, which encodes a tomato dehydrin, is triggered by osmotic stress and ABA . When over expressed in tomato, TAS14 confers partial drought and salinity tolerance . In our study, TAS14 increased rapidly after salt stress onset and remained elevated throughout the course of the stress treatment. Similar to NCED1, TAS14 did not show altered expression following P. capsici infection, and in the case of salt treatment, TAS14 expression returned to basal levels within 24 hpi .