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Phytophthora capsici is responsible for multiple disease syndromes of Capsicum annuum but the resistance mechanism is still unknown. Evaluating gene expression during foliar blight can be used to identify expression patterns associated with resistance in Capsicum species. This study reports a direct comparison of gene expression changes during the foliar blight syndrome using two different races of P. capsici on C. annuum host plants with resistant and susceptible phenotypes to those races. Four genes were evaluated for differential expression following leaf inoculation with P. capsici. RNA isolated from leaves at three time points was used to quantify gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Of four genes tested, two had differential expression in response to P. capsici at 72 hours postinoculation, a xyloglucan-specific endo-b-1,4-glucanase inhibitor protein (XEGIP2) in susceptible cultivar New Mexico Heritage 6-4 (NMH6-4), and a C. annuum cell wall protein (CWP) in resistant Criollo de Morelos 334 (CM334). Both genes had a 5-fold increase in transcription in leaves over the control. These results suggest that both genes are playing a role in disease resistance to foliar blight. Chile pepper (C. annuum L.) is an important vegetable crop grown worldwide, a rich source of dietary b-carotene. The capsaicin in the fruit is used for pain relief and the extracted pigments for use in cosmetics and foods. In the United States alone, chile pepper consumption has increased to an annual per capita of 16.4 pounds while the U.S. crop is 4.8 million cwt with a value of $146.8 million (Burden, 2014). One of the major challenges to chile pepper production is yield losses due to pathogen interactions, specifically the soilborne oomycete P. capsici (Leonian, 1922). No commercial variety of C. annuum has universal resistance to P. capsici (Walker and Bosland, 1999), but C. annuum landrace, CM334 has the highest level of resistance against all disease syndromes caused by P. capsici: i.e., root rot, foliar blight, fruit rot and, stem blight (Alcantara and Bosland, 1994; Walker and Bosland, 1999). To date, the genetic basis for resistance is not yet fully understood and the number of genes controlling resistance in CM334 remains unknown (Castro-Rocha et al., 2012; Rehrig et al., 2014). However, a multiple gene system for resistance has been proposed in the P. capsici–C. annuum pathosystem (Bnejdi et al., 2009; Lee et al., 2012; Monroy-Barbosa and Bosland, 2008; Walker and Bosland, 1999), and to further complicate the matter, variation in race or isolate of P. capsici, results in high genetic variability within the species (Truong et al., 2010) and different cultivars of chile pepper are resistant to one race but may be susceptible to another (Monroy-Barbosa and Bosland, 2008; Sy et al., 2005). Inoculum concentration also appears to have an effect on gene expression in CM334 (Castro-Rocha et al., 2012; Lee et al., 2012). The relationship between P. capsici and its host plants is complex, and more research is needed to determine the molecular bases for the resistance to these disease syndromes (Lamour et al., 2012; Thines and Kamoun, 2010). Regardless of the genetic mechanism of disease resistance, following exposure to a pathogen, most plants initiate gene expression changes (reviewed in Wise et al., 2007). Genes for proteins located in the plant cell wall and involved in plant defense mechanisms are often differentially expressed in response to many different types of pathogens (reviewed in H€uckelhoven, 2007). Also, transcripts for a large complex of structurally diverse gene products called pathogenesis related (PR) are increased following exposure to pathogens (Soh et al., 2012). PR gene products are modeled to contribute to an overall defensive condition (Sels et al., 2008; Van Loon and Van Strien, 1999). In previous work, we screened thousands of transcripts and identified 168 genes with differential expression patterns inC. annuum– P. capsici root rot syndrome (Richins et al., 2010). Among those genes was a gene for a XEGIP; referred to here as XEGIP2 (Genbank accession: EB084827, 99% similar to FJ606761). XEGIP2 has elevated expression in response to P. capsici (race PWB24) in root rot in both a resistant, CM334, and susceptible cultivar, NM6-4, of C. annuum (Richins et al., 2010). This gene is modeled to inhibit P. capsici produced xyloglucanspecific endo b-1,4 glucanase (XEG), which specifically attacks the xyloglucan bonds in plant cell walls (Yoshizawa et al., 2012) by hydrolyzing the xyloglucan and loosening the cross-linkages (Hayashi, 1989). XEGIPs inhibit XEGs from breaking down the xyloglucan in plant cell walls and provide some resistance against the pathogen (Jones, 2012; Scarafoni et al., 2010). XEGIPs have been identified in other members of the Solanaceae family such as potato (Solanum tuberosumL.), which has a complex XEGIP gene family with nine members in addition to the original XEGIP. Silencing of a member of this gene family resulted in increased susceptibility (Jones et al., 2006). In chile, a secondXEGIP, referred to here as XEGIP1, is modeled to play a role in the hypersensitive response (HR) associated with disease resistance to bacterial pathogens (Choi et al., 2013). This present study was designed to 1) determine if gene expression induced by pathogen challenge could be detected in leaf discs when the inoculum concentration was at levels used to assay for resistance phenotypes; 2) determine if the gene expression changes could distinguish resistant and susceptible hosts; and 3) determine if the gene expression changes could detect pathogen Received for publication 27 Apr. 2015. Accepted for publication 21 July 2015. This work was supported in part by the New Mexico Agricultural Station, and support from the U.S. Department of Agriculture–Agricultural Research Service through a Specific Cooperative Agreement. Corresponding author. E-mail: moconnel@nmsu. edu. 1342 HORTSCIENCE VOL. 50(9) SEPTEMBER 2015 race-specific responses. This study reports for the first time, a direct comparison of the gene expression changes during the foliar blight syndrome using two different races of P. capsici on C. annuum host plants with resistant and susceptible phenotypes to those races. Further, this study selected four genes for analysis whose expression in a root rot syndrome had already been characterized, thereby allowing a comparative discussion of these two disease syndromes. Of those four genes, the CWPwas first reported as a disease responsive gene in a screen for PR genes expressed during the HR ofC. annuum leaves to Tobacco mosaic virus (TMV) (Shin et al., 2001); the Universal Stress Protein, is similar to a bacterial stress responsive gene found throughout the bacterial, fungal, and plant kingdoms, with potential roles in biotic and abiotic stresses in plants (Kerk et al., 2003); and two were separate gene family members of the XEGIP class, i.e., XEGIP1 and XEGIP2. The genes for the Universal Stress Protein and the CWP were selected for investigation in this foliar blight study since transcripts for these genes increase in response to root rot challenge only in resistant genotypes of Capsicum (Richins et al., 2010). Materials and Methods Chile and P. capsici inoculation. Seeds of C. annuum L., CM334 and NMH6-4, were germinated in Metro Mix 360 Professional Growing Medium (Agawam, MA), for about 2 weeks at 28 ± 1 C in a growth chamber. Seedlings (36 of each cultivar) were then transferred to 10.16 · 10.16 cm plastic containers (T.O. Plastics, Minneapolis, MN) and planted in Metro Mix 360. Plants were maintained in a greenhouse until they reached the four to six true leaf stage. For foliar blight inoculation, two races of P. capsici were used, a virulent New Mexico isolate PWB24 (ATCC MYA 2289) race 1, mating type A1 (Sy et al., 2008), and a lesser virulent New Jersey isolate PWB54 (Hallock, ATCC MYA 2338) race 9, mating type A2 (Oelke et al., 2003). Phytophthora isolates were established on V8 agar (Tuite, 1967) following essentially the protocol ofMonroyBarbosa and Bosland (2010). Squares of agar-containing P. capsici were cut from the V8 plates and transferred to a new plate with enough distilled water to cover the squares. Plates were placed under a fluorescent light for 2 days to induce sporangia formation. Plates were then placed in a –10 C freezer for 10 min of cold shock to release the zoospores, and then transferred to a 29 C incubating oven for 30 min to allow recovery. A hemocytometer (Hausser Scientific BrightLine Hemacytometer, VWR, Radnor PA) was used to calculate the concentration of zoospores and stock solutions were adjusted with distilled water to achieve a final concentration of 40,000 zoospores/mL. Plants in their pots were placed in one of three 99-L plastic bins: 81.6 · 48.6 · 34.9 cm (Sterilite, Townsend, MA); adding 500 mL of hot (53 C) water to the bins created a humid environment. A 0.5 cm disc of germination paper (Nasco, Modesto, CA) was placed on one leaf per plant and 50 mL of inoculum per leaf was dispensed using a micropipette. Control plants received 50 mL of distilled water on the disc. Experimental and statistical design. A complete randomized split plot design was used and two complete replications of the experiments were carried out. Each replication, called ‘‘trial’’ contained two experiments, exposure of both cultivars to one race (#1 PWB24), and then separately to the other race (#2 PWB54). Then repeated for the second trial, exposure to one race (#3 PWB24), and again separately to the other (#4 PWB54) for a total of four experiments in Fig. 1. Leaf tissue collection for RNA extraction. Leaf removed from plant, 2.8-cm diameter disc surrounding inoculation site was cut and weighed. Table 1. Primer sequences used for qRT-PCR analysis. Gene Primer sequences Annealing

Detection of Gene Expression Changes in Capsicum annuum L. Foliar Blight Caused by Phytophthora capsici Leon. Using qRT-PCR and Leaf Discs