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, but remained in a predisposed state in the absence of visible stress FIGURE 4 | ABA-related gene expression in inoculated ‘New Yorker’ tomato seedling roots following an episode of salt stress. NCED1 and TAS14 expression in non-stressed roots and in roots after 18 h exposure to 0.2 M NaCl/0.02 CaCl2, with and without inoculum . NCED1 and TAS14 expression was normalized against Cyp and UK. Values are the means ± SE from two experiments, with roots from fifive plants pooled per sample and six samples analyzed for each treatment mean per time point. Note that seedlings were removed from the salt stress and returned to 0.5X Hoaglands during the course of analysis. symptoms for up to 24 h following removal from the salt. The salt stress effect on disease appears to operate through an ABAdependent 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,hydroponic vertical farm exudates from salt-stressed roots of wild-type tomato plants and ABA-defificient 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 FIGURE 6 | P. capsici colonization 48 hpi on ‘New Yorker’ and NahG tomato seedlings non-stressed or salt stressed with 0.2 M NaCl/0.02 M CaCl2 for 18 h. Colonization estimated by qPCR of pathogen DNA. Letters indicate significant differences at P = 0.05 . Values are the means ± SE from three experiments, with five samples, each from a separate seedling, for each treatment within each experiment . 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 biotrophy 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 NCED3is 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 topre-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 . The possibility of P. capsici-derived ABA was of interest because some plant pathogenic fungi produce ABA , and some stramenopiles such as the malarial pathogen, Plasmodium falciparum, are capable of ABA synthesis . However, we did not detect ABA in P. capsici culture filtrates or mycelium by immunoassay , and genes encoding the necessary biosynthetic enzymes are not evident in oomycete genomes . Furthermore, we found no evidence that P. capsici infection further engages the pathway as part of its infection strategy, either in non-stressed or salt-stressed tomato plants.

These results indicate that salt stress, but not Phytophthora infection, strongly engages the ABA pathway in tomato roots – NCED1 and TAS14 gene expression, and ABA synthesis and accumulation. The SA-induced tomato PR protein, P4, is homologous to PR-1 in tobacco and Arabidopsis. P4 gene expression is induced in tomato leaves by plant activators , pathogens, including Phytophthora infestans, and the oomycete elicitor arachidonic acid . We found that infection of tomato roots by P. capsici strongly induces P4, but exposure of the roots to salt prior to inoculation essentially abolished P4 expression relative to non-stressed, inoculated plants . Similarly, expression of the JA-induced PI-2 was significantly reduced in infected plants that had been previously salt-stressed. Our findings that salt stress prevents pathogen-induced SA- and JA-regulated gene expression are consistent with results in other plant–microbe interactions that demonstrate ABA-mediated suppression of SA and JA defense responses . Tomato plants suppressed in SA accumulation by the nahG transgene are more susceptible to P. capsici than the wild type control plants in both non-stressed and salt-stressed assay formats . This suggests a role for SA-mediated responses in partially limiting P. capsici colonization. However, the proportional increase in pathogen colonization observed in salt-stressed plants relative to non-stressed plants is the same in both WT and NahG backgrounds. Impairment of SA action by salt stress may contribute to increased pathogen colonization; however, we did not see a compounding effect of the SA-deficiency in NahG plants on stress-induced disease severity. Salicylic acid’s role in tomato resistance to P. capsici is complex. In a study using chemical activators that mimic SA action to induce resistance, we found these activators when applied to roots induced systemic protection of tomato leaves against the bacterial speck pathogen , with and without predisposing salt stress . However, these same plant activator treatments afforded no protection against P. capsici, with or without the salt stress treatment. Pst and P. capsici are quite different in their infection strategies and requirements, as well as the organs they attack in the plant, so interpreting differences in disease outcomes following different treatments is a speculative exercise, at best. P. capsici may simply be a more aggressive pathogen relative to Pst,vertical farm and our experimental format is highly conducive to root and crown rot disease. So P. capsici attack overwhelms any chemically induced resistance that is otherwise capable of withstanding Pst challenge. NahG expression may impair a set of SA-mediated defenses that are effective against P. capsici, but differ from a subset, induced by chemical activators, that are insufficient to resist this pathogen. The JA-deficient tomato mutants acx1 and def1 in the ‘Castlemart’ background are compromised in defense against insects and pathogens . Although severity of the predisposition phenotype can vary among tomato cultivars, we were astonished that ‘Castlemart’ and its JA mutants were not predisposed by salt, strongly trending instead toward enhanced resistance . This suggests a stress response in ‘Castlemart’ that is different from other tomato genotypes we have examined in predisposition studies. The reason for this is unclear, and limited resources precluded our further examining predisposition in this cultivar. Unlike the other genotypes used in our study, ‘Castlemart’ is a processing variety with a pedigree that may have incorporated different stress tolerances. It is a determinate variety that was bred for arid climates, and arid zone soils are more commonly associated with salinity . ‘Castlemart’ has been reported to accumulate proteinase inhibitors in response to high salinity . Jasmonic acid and its methyl ester when applied to leaves can induce resistance in tomato to P. infestans . Arabidopsis mutants in JA perception and synthesis are more susceptible to oomycete pathogens. Studies with other oomycete diseases also illustrate JA’s importance in resistance .

We found that exogenous JA enabled tomato roots to respond in a manner that partially offffset the salt stress impairment of PR-protein gene expression . The induction of P4 only during infection of JA-treated plants is reminiscent of the reported sensitization by methyl jasmonate of the plant’s response to eicosapolyenoic acid elicitors released during infection by Phytophthora species and potentiation of JA signaling by the plant activator β-aminobutryic acid . Our results with the tomato genotypes and treatments used in this and previous studies affirms ABA’s dominant effect relative to the salt-induced impacts on SA and JA action during predisposition to Phytophthora root and crown rot. ABA appears to be necessary to predispose tomato seedlings to this disease following acute salt stress. However, results presented here and previously indicate that priming through chemical activation of the SA and JA response networks may partially offset the stress-induced impairment of defense-related gene expression and the increased susceptibility in tomato to certain pathogens. We recognize that the response pathways modulated by ABA, JA and SA during episodic root stress may interact in subtle ways beyond the resolution afforded by the pathosystem and treatments we selected . Comparative transcriptomics, proteomics and metabolomics of plants under predisposing stress should help identify key regulatory features . Studies with additional mutants as well as salt- and drought-tolerant genotypes also may reveal additional variation that could be useful to refine our understanding of the abiotic-biotic stress ‘interactome’ . This information could suggest novel targets to mitigate the impact of root stresses that increase severity of soil borne diseases. Inorganic pyrophosphate is an intermediate compound generated by a wide range of metabolic processes, including biosynthesis of various macro-molecules such as proteins, DNA, RNA, and polysaccharides. Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion.