agronomy Elicitor Induced JA-Signaling Genes Are Associated with Partial Tolerance to Hemibiotrophic Pathogen Phytophthora capsici in Capsicum chinense

: Phytophthora capsici causes root and stem rot disease in Capsicum. However, molecular mechanisms underlying this pathosystem are little known. The use of elicitors as tools that trigger defense responses to biotic stresses to study molecular plant defense has increased. In this study, early defense induced in the susceptible cultivar C. chinense using three elicitors to assess its role during interaction with hemibiotrophic P. capsici . The response to infection by phenotypic analyses across the time during disease development in seedlings treated with elicitors was compared. Likewise; defense-gene expression were investigated by qRT-PCR. A total of ﬁve resistance genes were used as markers of signaling pathways mediated by jasmonate/ethylene (JA/ET) and salicylic acid (SA). Further, six R genes analogs (CcRGAS) related to oomycete-defense were employed. The results showed that elicitors MeJA and b-aminobutyric acid (BABA) slightly reduced disease symptoms. Moreover, MeJA or BABA treatments followed by challenge with P. capsici up-regulated the expression level of genes related to the JA/ET signaling pathway ( CcLOX2 , CcPDF1 and CcETR1 ). Furthermore, MeJA treatment followed by challenge triggered a signiﬁcant induction of de CcRGAS and CcRPP13 expression within 24 h of inoculation. This suggests that in the early defense mechanisms against P. capsici JA signaling plays an important role.


Introduction
The genus Capsicum L. (Capsiceae, Solanaceae), comprises 42 species, five of great economic importance (C. annuum var. annuum, C. chinense, C. frutescens, C. baccatum varieties pendulum and umbilicatum, and C. pubescens) were domesticated by American natives [1][2][3]. It has been reported that, the Yucatan Peninsula, Mexico, has a large number of Capsicum haplotypes, many of which are unique, suggesting an important region of chili domestication and center of diversity. The pepper species most commonly grown in Yucatan Peninsula, is C. chinense (Habanero) whose varieties have as characteristic its high pungency [4][5][6].
Phytophthora capsici is a hemibiotrophic soil-borne oomycete with two distinct stages of infection. An initial biotrophic infection phase, where the cells do not appear to be affected, which is followed by a second necrotrophic phase, killing infected cells and causing significant tissue collapse and necrosis [7]. P. capsici was first described in New Mexico in 1922 causing severe disease symptoms such as foliar blight, stem blight, and root,

Plant Material
C. chinense genotype Mayan Ba alché maintained at the Centro de Investigación Científica de Yucatán, México (provided by PhD Santana-Buzzy), was used in this study. In vitro culture was performed according to Núñez-Pastrana et al. (2011) with slight modifications. C. chinense seeds were surface sterilized with 70% ethanol for 5 min, rinsed three times with sterile distilled water, washed with sodium hypochlorite (1.6%) for 40 min, followed by a second three-time rinse with sterile distilled water and finally incubation in 40 mL sterile water with 250 µg mL −1 of chloramphenicol. Seeds were transferred to cotton and water in sterile conditions. Seeds with the radicle emerged were transferred to Magenta boxes containing Murashige and Skoog medium salts (Sigma, Saint Louis, MA, USA) supplemented with vitamins and carbon source. Finally, C chinense seed were grown for four week.

Inoculation Method
The virulent strain of P. capsici (CPV-279, provided by PhD Sylvia Fernández-Pavía, Universidad Michoacán de San Nicolás de Hidalgo) was grown in petri dishes on potato dextrose agar medium in dark conditions. From each plate that was completely covered by the mycelium, one mm-diameter plug was aseptically transferred to the center of a new PDA plate, after six days of growth, mycelium was used to inoculate pepper seedlings. Four-week old pepper seedlings were inoculated in the adaxial surface of the third and fourth true leaves with a 1 mm-diameter plug. Subsequent to P. capsici inoculation, disease development was assessed using a disease rating scale from 0-5 according to Monroy-Barbosa and Bosland (2010) [24] with minor modifications, in which 0 = No disease symptoms, 1 = Water soaked, 2 = area in contact with pathogen was necrotized, 3 = inoculated leaves were necrotized, 4= plant stem was necrotized and mycelium was growing, and 5 = systemic leaves were necrotized. The score of plants were recorded following across the time during disease development.
Disease index percentages were recorded based on the following formula according to Chakraborty

Treatment of Seedlings
The elicitor molecules used in the experiment were; 150 µM SA, 100 µM MeJa, and 1 mM BABA (Sigma, Saint Louis, USA). The elicitor molecular were used in independently trataments. SA and BABA were dissolved in H 2 O; and MeJA was dissolved in 0.1% Triton X 100. Different sets of treatments were carried out with the four-week old seedlings. In the first set, the seedlings were sprayed with SA, MeJA, and BABA, along with water or Triton X 100, these last two were maintained as control in parallel. The treated seedlings were harvested after 24 h for RNA extraction.
The second set of seedlings treated with the elicitor molecules as mentioned above and 24 h after were inoculated with P. capsici. This second group was subdivided into two subgroups. For the assessment of disease development, a representative group of plants was monitored daily for 72 h post inoculation (hpi) using the disease severity scale described above. For differential expression analysis, leaves from 24 hpi with P. capsici and from mockinoculated (Water or Triton X 100 alone) plants were collected and stored at −0 • C.

RNA Extraction
RNA was extracted from the leaves of both mock and inoculated pepper seedlings. PureZOL RNA isolation reagent (Bio-Rad, Hercules, CA, USA) was used to extract the total RNA from samples according to the manufacturer's specifications. Extracted RNA was quantified with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For quantitative real-time PCR (qRT-PCR) analysis, RNA samples were treated with DNase I (1 U µL −1 , Thermo Fisher Scientific, Waltham, MA, USA). Total RNA was stored at −80 • C until qRT-PCR assay.

Expression Analysis through qRT-PCR
Total RNA was used for qRT-PCR performed according to the standard Advanced cDNA Synthesis Kit for RTqPCR (Bio-Rad, Hercules, CA, USA), and the iTaq™ Universal SYBR ® Green Supermix (Bio-Rad, Hercules, CA, USA). The qPCR conditions were those recommended by the manufacturer: one cycle of pre-treatment at 50 • C for 2 min, one cycle at 95 • C for 10 min, and 40 cycles at 95 • C for 15 s and at 60 • C for 1 min. The results presented are from three independent biological replicates. Each biological replicate was tested by triplicate and data were normalized to GADPH reference gene for Capsicum. The genes selected for analysis were from two main groups; defense marker genes related to the signaling pathway and R genes encode for NBS-LRR (CcRGAs).
The first group included the master regulator CcNPR1, genes involved in response to biotic stress as CcLOX2 (JA synthesis) and CcETR1 (ET responsive genes), just like genes encoding pathogenesis related proteins, like CcPR4, and a defense responsive gene (CcPDF1, encoding a plant defensin). In group two were selected CcRGA18, CcRGA23, CcRGA36, and CcRGA38 genes that have been reported with a high degree of similarity with the R3a-like disease-resistance protein gene from Solanum demissum, additional, was included CcRGA2 and CcRPP13, all these genes have been reported as involved in defense mechanism to oomycete Phytophthora [15,16,27,28].
Appropriate primers for each gene were employed for qRT-PCR ( Table 1). The 2 −∆∆CT method was used for relative quantification, where the ∆∆CT value = ((C T gene of interest − C T internal control) sample A − (C T gene of interest − C T internal control) sample B) [29]; where sample A corresponds to plants with treatment and sample B corresponds to mock inoculated plants.

Statistical Analysis
Statistical significance was determined with multiple Student's t-test, followed by the Holm-Šídák multiple comparison test at a significance value of 0.05, by using the Ghaph-Pad Prism (6.0, GraphPad Software, San Diego, CA, USA, http://www.graphpa.com Accessed date: 21 March 2013). All the experiments, with at least three biological replicates, were conducted and evaluated separately.

MeJA and BABA Treatments Modifies the Response of C. chinense Plants by Reducing Severity of Disease Symptoms
The capacity of externally applied elicitors to induce tolerance has been documented. However, this response depends on the type of pathogen. To assess the effect of the elicitor molecules on the interactions of C. chinense with the pathogen P. capsici, plants were treated with selected elicitors (150 µM SA, 100 µM MeJa, and 1mM BABA), and 24 h after were challenged with the oomycete.
In non-treated seedlings of the susceptible species C. chinense, P. capsici rapidly infected the plant. In the inoculated leaves at 24 hpi was observed a small lesion with water-soaked appearance under the area in contact with the mycelia (

Effects of Elicitor Application in the Expression of Defense-Related Genes
We investigated the effect of exogenous application of 150 μM SA, 100 μM MeJa, and 1 mM BABA in the expression levels of some defense-related genes in C. chinense. Differences in the expression of defense marker genes among treatments confirmed that exogenous application of the corresponding elicitor molecules stimulated the corresponding signaling which is under its modulation ( Figure 3).

Effects of Elicitor Application in the Expression of Defense-Related Genes
We investigated the effect of exogenous application of 150 µM SA, 100 µM MeJa, and 1 mM BABA in the expression levels of some defense-related genes in C. chinense. Differences in the expression of defense marker genes among treatments confirmed that exogenous application of the corresponding elicitor molecules stimulated the corresponding signaling which is under its modulation ( Figure 3).

Effects of Elicitor Application in the Expression of Defense-Related Genes
We investigated the effect of exogenous application of 150 μM SA, 100 μM MeJa, and 1 mM BABA in the expression levels of some defense-related genes in C. chinense. Differences in the expression of defense marker genes among treatments confirmed that exogenous application of the corresponding elicitor molecules stimulated the corresponding signaling which is under its modulation ( Figure 3).  At 24 h after MeJA treatment a significant increase in expression (p < 0.05) of the JA biosynthetic gene CcLOX2 (1.9-fold change due to treatment) and CcPR4 gene (2.24-fold change due to treatment), was observed, while transcripts of CcPDF1 (−1-fold) and CcNPR1 (−1.53-fold) showed down-regulation in JA-treated plants compared to control plants, indicating an effect of MeJA in the signaling process (Figure 3a,d-f).
As expected, SA treatment led to the activation of the CcPR4 gene (2.3-fold), however (Figure 3f). Although, CcNPR1 transcript did not show significant differential expression after 24 h treatment, the antagonistic effect of SA on methyl jasmonate-induced CcLOX2 (−1.61-fold) gene It were observed (Figure 3a,d).
In addition, the effect of non-protein amino acid BABA in the expression of other defense responsive genes was analyzed. Genes encoding CcETR1 (1.68-fold), and CcPDF1 (2.045-fold), showed a significantly higher relative expression (p < 0.05) only in BABAtreated plants, just like the other elicitors, BABA induced the expression of CcPR4 (2.31-fold) (Figure 3c,e,f). At 24 h after MeJA treatment a significant increase in expression (p < 0.05) of the JA biosynthetic gene CcLOX2 (1.9-fold change due to treatment) and CcPR4 gene (2.24-fold change due to treatment), was observed, while transcripts of CcPDF1 (−1-fold) and CcNPR1 (−1.53-fold) showed down-regulation in JA-treated plants compared to control plants, indicating an effect of MeJA in the signaling process (Figure 3a,d-f).

Increased Expression of Genes
As expected, SA treatment led to the activation of the CcPR4 gene (2.3-fold), however (Figure 3f). Although, CcNPR1 transcript did not show significant differential expression after 24 h treatment, the antagonistic effect of SA on methyl jasmonate-induced CcLOX2 (−1.61-fold) gene It were observed (Figure 3a,d).
In addition, the effect of non-protein amino acid BABA in the expression of other defense responsive genes was analyzed. Genes encoding CcETR1 (1.68-fold), and CcPDF1 (2.045-fold), showed a significantly higher relative expression (p < 0.05) only in BABAtreated plants, just like the other elicitors, BABA induced the expression of CcPR4 (2.31fold) (Figure 3c,e,f).

Increased Expression of Genes Associated with JA-ET Signaling Is Associated with C. chinense Partial Tolerance to P. capsici
We analyzed the expression of genes associated with P. capsici tolerance in plants previously treated with chemical elicitors. The transcript levels of the genes were assessed for determining the effectiveness of the defense induction of chemical elicitor 48 h after treatment. The expression of CcNPR1, CcETR1, CcLOX2, CcPDF1, and PR4 were induced with certain treatments compared with control (p < 0.05) ( Figure 4); SA induced CcNPR1   The expression levels in plants infected with P. capsici, showed that expression of CcPR4 (11.43-fold), and CcPDF1 (1.67-fold) increased (Figure 4d,e). The rest of the analyzed genes did not significantly change at the transcript level at 24 hpi. Meanwhile, a magnified response was observed when BABA elicitor treatment was coupled with P. capsici infection inducing gene expression of CcPDF1 (6.68-fold), and CcETR1 (1.58-fold) (Figure 4b,e). Similar results were observed in CcLOX2 (2.37-fold), since its expression increased in response to MeJA + P. capsici (Figure 4c). The expression of CcPR4 (10.64-fold) was induced in SA + P. capsici in similar levels as the simple infection (11.43-fold), while CcETR1 (2-fold) expression was boosted at similar levels to only SA treatment (1.44-fold) (Figure 4b,d).

Expression of NBS-LRR Encoding R Genes in Response to Elicitor Treatment and P. capsici
In the compatible interactions has been reported that the expression of ARG is at a relatively low-level. However, treatment with chemical elicitors effectively up-regulates the expression of this genes in a susceptible host [15,23]. The expression levels in plants infected with P. capsici, showed that expression of CcPR4 (11.43-fold), and CcPDF1 (1.67-fold) increased (Figure 4d,e). The rest of the analyzed genes did not significantly change at the transcript level at 24 hpi. Meanwhile, a magnified response was observed when BABA elicitor treatment was coupled with P. capsici infection inducing gene expression of CcPDF1 (6.68-fold), and CcETR1 (1.58-fold) (Figure 4b,e). Similar results were observed in CcLOX2 (2.37-fold), since its expression increased in response to MeJA + P. capsici (Figure 4c). The expression of CcPR4 (10.64-fold) was induced in SA + P. capsici in similar levels as the simple infection (11.43-fold), while CcETR1 (2-fold) expression was boosted at similar levels to only SA treatment (1.44-fold) (Figure 4b,d).

Expression of NBS-LRR Encoding R Genes in Response to Elicitor Treatment and P. capsici
In the compatible interactions has been reported that the expression of ARG is at a relatively low-level. However, treatment with chemical elicitors effectively up-regulates the expression of this genes in a susceptible host [15,23].  When infection was coupled to elicitor treatment the expression level of most of CcRGA genes was maximized; in MeJA + P. capsici treatment, CcRGA18 (29.12-fold), CcRGA38 (5.11-fold), CcRGA2 (2-fold), and CcRPP13 (5.88-fold) expression was increased; meanwhile in BABA + P. capsici treatment, only CcRPP13 (3.1-fold) gene was overexpressed. Interestingly SA + P. capsici treatment did not change significantly the expression of any of the evaluated genes ( Figure 5). When infection was coupled to elicitor treatment the expression level of most of CcRGA genes was maximized; in MeJA + P. capsici treatment, CcRGA18 (29.12-fold), CcRGA38 (5.11fold), CcRGA2 (2-fold), and CcRPP13 (5.88-fold) expression was increased; meanwhile in BABA + P. capsici treatment, only CcRPP13 (3.1-fold) gene was overexpressed. Interestingly SA + P. capsici treatment did not change significantly the expression of any of the evaluated genes ( Figure 5).

Discussion
Early defense activation is important during the interaction with different pathogen. Depending on the infection strategy and lifestyle of invading pathogens, several evidence show a correlation between the timing response and the outcome for plant resistance or susceptibility. Phytophthora species are among the most destructive plant pathogens with hemibiotrophic lifestyles. P. capsici is an important pathogen of Solanaceae, being one of the most devastating pathogens in pepper production worldwide [7]. On the other hand, elicitor molecules can modulate the activation of defense pathways, several of these induce resistance to hemibiotrophic pathogens. In the current work, we investigated the effect of elicitor molecules in restricting the infection of hemibiotrophic pathogen P. capsici, on C. chinense seedlings. Moreover, we explored the relationship between the slight tolerance granted by some of the elicitor molecules and the regulation of defense signaling pathways by means of analyzing differential expression of defense-marker genes and R genes, at the early time of infection.
In the present study was observed, the transitional phase to necrotrophy at 24 hpi with the presence of the symptom of water soaking, followed by the beginning of necrosis at 48 hpi (Figure 1). This phenotype is characteristic in hemibiotrophic interactions, where the pathogen switches its strategy of infection (biotrophy-to-necrotrophy), in P. capsici this interphase appears between 24 and 48 hpi [7,34,35]. We mimic early plant defenses using chemical inducers, specifically SA, MeJA and BABA. We found that MeJA and BABA treatments resulted in slightly enhanced tolerance in C. chinense against P. capsici by reducing the severity of symptoms during the infection cycle. For instance, we observed that in MeJA treated seedlings and inoculated with P. capsici, symptoms of necrosis occurred at a later time (Figures 1m and 2b). On the other hand, although the infection process of P. capsici could not be completely abolished by 1 mM BABA, its application produced a modification of the disease symptoms, observing these after 48 hpi (Figures 1g-i and 2b). These results are consistent with previous reports, where BABA treatment caused inhibition of disease development in various host-pathogen interactions including pathogens of the genus Phytophthora, which are highly destructive in the necrotrophic phase of the infection cycle [36][37][38]. Further, the effects against hemibiotrophic pathogens by reducing necrotrophic lesions by MeJA application have been observed in Camellia, Sesame and Potato [39][40][41]. On the other hand, the activation of the response in a timely manner is very important for plant disease resistance [40]. We observed a protective effect in MeJA and BABA treated plants against P. capsici probably achieved by the activation of defense mechanisms modulated by the phytohormone that counteracts the pathogen at that point of infection.
Here, we focused on providing data on the expression of marker genes of the signaling pathways after elicitor treatment and before infection. We analyzed the difference in expression of two groups of genes at 24 h after treatments: (i) genes induced by SA signaling pathways (NPR1 and PR4); and (ii) genes induced by JA (LOX2, PDF1.2, ETR1, and PR4). Our results showed that MeJA and BABA pretreatments were effective in increasing the expression levels of genes related to the JA-mediated pathway such as LOX2, PDF1, and ETR1 (Figure 3). The Arabidopis thaliana genome encodes multiple 13-lipoxygenases (13-LOXs) involved in the biosynthesis of jasmonate precursors. In pepper CaLOX2 is classified as 13-LOX, exogenous JA application induce its expression. Further, LOX2 can be stimulated under responses to hemibiotrophic pathogens [40][41][42][43]. On the other hand, Plant Defensin type 1 genes (PDF1s) are considered markers of JA activation signaling cascade, are usually associated with the response to necrotrophic and hemibiotrophic pathogens following activation of ET and JA [39,44]. This results verifies the early defense activation by JA pathways in MeJA and BABA treatments.
The completely different phenotypes after infection in resistant or susceptible plants depend on complex signaling mechanisms to initiate a defense response after pathogen recognition. It has been shown that resistant and susceptible cultivars have different transcriptional responses to the challenge, the rapid and strong up-regulation of defense genes is observed in resistant varieties in contrast with a basal level or down-regulation in the susceptible ones which present severe symptoms [40,45]. Duo to the above, in this study we analyzed the difference in expression of the two groups of genes above and add a third (iii) R-genes which mediate plant defense responses (RGAs and RPP13). Because several authors hypothesize that plant resistance is effective during the early time of infection in the asymptomatic phase or at the inter-phase for hemibiotrophic pathogens [40], we analyzed at an early time after infection (24 hpi with water soaking symptoms). Most genes analyzed remained at basal levels in response to P. capsici at the early time of infection (24 hpi), as shown by the qPCR results. However, genes like CcPR4, and CcPDF1.2 were up-regulated (Figure 4). These results are consistent with those reported in compatible interactions, it is noted a lower expression of defense genes in the presence of the pathogen compared to its resistant counterpart. In tomato during a compatible interaction with P. infestans, transcript abundance of some genes was substantially reduced during the biotrophic phase (early times of infection, 48 hpi). On the other hand, it has been reported that some defense gene activation may occur in the early time after infection; example of this were reported in compatible interactions of P. infestans in potato, P. parasitica in Arabidopsis, and P. medicaginis in chickpea, where it was shown that PR4 gene is induced in early times of infection [35,46,47]. In the case of PDF1 gene, it was induced in the compatible interaction between C. chinense and P. capsici [20]. Additionally, we analyzed the expression of some R genes. In this study, the expression pattern of all RGAs evaluated in C. chinense during P. capsici infection was down-regulated ( Figure 4). Our data are similar to observed in qPCR analysis by Veena et al. (2016), that revealed down-regulation of RGA transcripts in a susceptible cultivar of pear millet (Pennisetum glaucum) following inoculation with Sclerospora graminicola [23]. The plant R genes encode NBS-LRR proteins that recognize effector proteins (Avr) from pathogens. Several R genes confer resistance to members of the Phytophthora genus, for example R3A is related to resistance to P. infestans in potato, RPP13 confers resistance to the oomycete pathogen Peronospora parasitica in Arabidopsis thaliana. Furthermore, it has been reported that CaRGA2 gene of C. annuum CM334 may participate in the resistance response against P. capsici [16,28,48]. Moreover, there is increasing evidence on how Phytophthora effectors manipulate host immunity [10]. Considering this, in this compatible interaction, P. capsici could be able to overcome or suppress the plant defense response within the first 24 hpi. According to our results, it seems that C. chinense recognizes P. capsici and activates a defense-related mechanism. However, it is not strong enough to counteract the infection, possibly due to a change of strategy of the pathogen.
The CcETR1, CcPDF1.2 and CcLOX2 genes upon treatments with MeJA are capable of increase their expression with P. capsici inoculation, treatment with BABA had this effect on the transcripts of CcPDF1.2 gene (Figure 4). These expression profiles have been observed in the priming induced by elicitors in other plants against pathogens [38,[49][50][51]. Interestingly, the genes affected in this way in the MeJA and BABA treatments in the present study, are those related to the response to JA/ET. In hemibiotrophic-pathogen host interaction, SA and JA pathways defines plant defense strategies [9,40]. Added to this evidence, recent studies report that exogenous MeJA application provides resistance against hemibiotrophic pathogens by activating JA signaling in potato and camellia [40,41]. This verifies that early defense activation by JA pathways could be contributing to P. capsici tolerance at this point in the early infection.
On the other hand, R-genes may be induced when an unusual perturbation occurs, some are up-regulated in response to exogenous application of chemical elicitors [23,52]. In the present study, CcRGA2 and CcRPP13 genes, increased their expression in response to MeJA treatment. Interestingly, this over-regulation was potentiated by P. capsici inoculation ( Figure 5). These transcript profiles were observed with CaRGA18 and CaRGA38, which have a high degree of similarity with R3a-like disease-resistance protein gene from Solanum demissum. In the case of BABA treatment, only CcRPP13 presented these transcript profiles. These data are consistent with what was observed in Arabidopsis where previous exposure to cold shock led to a significant increase in resistance to P. syringae pv. AvrPphB2. The authors propose that the environment can prime disease resistance through up-regulation of R-genes for preparatory response to conditions that alter the probability of invasion by pathogens [52]. The high level of CaRGA transcript associated with resistance to oomycetes in Solanaceae, suggests that it must be contributing to the partial tolerance observed in C. chinense against P. capsici.

Conclusions
JA and BABA contribute to the partial tolerance of habanero pepper (C. chinense) to P. capsici, these results indicate that JA and its signaling pathway might be working in the early stages of the interaction. However, P. capsici can counterattack this defense. Given this, we conclude that early induction and high intensity of defense modulated by the correct pathway for the corresponding phase of infection can confer tolerance. However, a hemibiotrophic pathogen is capable of counteracting that defense by changing the infection strategy.