Physiological responses of resistant and susceptible pepper plants to exogenous proline application under Phytophthora capsici stress

Phytophthora capsici Leon. is the main pathogen that limits the production of peppers. In this study, the effects of 1 and 10 mM proline (Pro), prior to exposure of resistant (CM-334) and susceptible (SD-8) pepper seedlings to P. capsici, on some physiological parameters were investigated. A lower Pro concentration (1 mM) was found to be more effective than 10 mM Pro in increasing the stress tolerance of the CM-334 cultivar. Namely, in CM-334 cultivar, the highest chlorophyll a, chlorophyll b, carotenoid, glucose and fructose content and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity percentage were detected on the seventh day after application of 1 mM Pro + P. capsici, while the lowest malondialdehyde (MDA) amount was measured on the third day in the same treatment. The highest ferric reducing antioxidant power (FRAP) increase was determined on the seventh day in the 10 mM Pro + P. capsici application. The effects of the same Pro treatments on the SD-8 cultivar somewhat differed; the highest amounts of chlorophyll a, chlorophyll b, anthocyanins, fructose, total protein and endogenous Pro were detected on the seventh day in the 1 mM Pro + P. capsici application, while the lowest MDA amount was measured on the third day after the 10 mM Pro + P. capsici application, the highest DPPH % and FRAP values were detected on the seventh day with 10 mM Pro + P. capsici application. Although some differences were detected between the cultivars, Pro application against the P. capsici stress in general resulted in a positive effect on photosynthetic pigments, soluble carbohydrates and antioxidant capacity in pepper. The exogenous application of Pro helped the non-resistant cultivar to overcome the stress.

Improving the Yield of Xenocoumacin 1 by PBAD Promoter Replacement in Xenorhabdus nematophila CB6

Xenocoumacin 1 (Xcn1), which is produced by Xenorhabdus nematophila CB6, exhibits strong inhibition activity against plant pathogens, especially fungi and oomycetes. Therefore, it has attracted interest in developing it into a novel biofungicide applicable for plant protection. However, its low yield with concomitant high cost during the fermentation process limits its widespread application. In this study, we replaced the native promoter of xcnA with the arabinose-inducible araBAD promoter (PBAD), a well-known and widely used promoter for expressing heterologous genes, to evaluate its effects on Xcn1 yield and antimicrobial activity. Compared with wildtype strain, the fermentation yield of Xcn1 was improved from 68.5 mg/L to 249.7 mg/L (3.6-fold) and 234.9 mg/L (3.4-fold) at 0.5% and 1.0% L-arabinose concentration, respectively. We further explored the transcription level of the biosynthesis related genes of Xcn1 and found that their upregulation resulted in the yield improvement of Xcn1. Moreover, the antimicrobial activity of Xcn1 against Bacillus subtilis and Phytophthora capsici was determined by agar diffusion plate and growth inhibition assay, as expected, it was also found to be enhanced. The promoter-replacement strategy utilized here improves the yield of Xcn1 efficiently, which provides a basis for the industrial production of Xcn1.

Gene Expression in Cucurbita spp. Root and Crown during Phytophthora capsici Infection

Phytophtora capsici causes major diseases in cucurbit crops worldwide. In this study, we inoculated this pathogen into Cucurbita pepo subsp. pepo susceptible MUCU-16 and C. moschata tolerant M63. The gene expression of plant pathogenesis-related proteins chitinase (CpChiIV), lignin-forming peroxidase (CpLPOX), and defensin (CpDEF) and hormone-related enzymes salicylic acid (CpPAL) and ethylene (CpACO) was analyzed for two weeks post-inoculation in root and crown tissues. Differentially expressed genes were found between genotypes, tissues, days post-inoculation, and inoculated/non-inoculated samples. After inoculation, CpPAL and CpChiIV (crown) were downregulated in MUCU-16, while CpLPOX and CpDEF were upregulated in M63. In inoculated samples, higher expression changes were presented on days 10–14 than on day 3 for CpACO, CpLPOX, and CpDEF genes. Overexpression was higher for CpDEF compared to the other tested genes, indicating good suitability as a marker of biotic stress. The overexpression of CpDEF was higher in crown than in roots for both inoculated genotypes. The basal expression of CpPAL and CpDEF was higher in MUCU-16, but after inoculation, CpPAL and CpDEF gene expression were higher in M63. These changes suggest an association between CpDEF upregulation and tolerance, and between CpPAL downregulation and susceptibility.

Quantitative genetic analysis of interactions in the pepper-Phytophthora capsici pathosystem

ABSTRACTThe development of pepper cultivars with durable resistance to the oomycete Phytophthora capsici has been challenging due to differential interactions between the species that allow certain pathogen isolates to cause disease on otherwise resistant host genotypes. Currently, little is known about the pathogen genes that are involved in these interactions. To investigate the genetic basis of P. capsici virulence on individual pepper genotypes, we inoculated sixteen pepper accessions – representing commercial varieties, sources of resistance, and host differentials – with 117 isolates of P. capsici, for a total of 1,864 host-pathogen combinations. Analysis of disease outcomes revealed a significant effect of inter-species genotype-by-genotype interactions, although these interactions were quantitative rather than qualitative in scale. Isolates were classified into five pathogen subpopulations, as determined by their genotypes at over 60,000 single-nucleotide polymorphisms (SNPs). While absolute virulence levels on certain pepper accessions significantly differed between subpopulations, a multivariate phenotype reflecting relative virulence levels on certain pepper genotypes compared to others showed the strongest association with pathogen subpopulation. A genome-wide association study (GWAS) identified four pathogen loci significantly associated with virulence, two of which colocalized with putative RXLR effector genes and another with a polygalacturonase gene cluster. All four loci appeared to represent broad-spectrum virulence genes, as significant SNPs demonstrated consistent effects regardless of the host genotype tested. Host genotype-specific virulence variants in P. capsici may be difficult to map via GWAS, perhaps controlled by many genes of small effect or by multiple alleles that have arisen independently at the same loci.

Identification and functional deciphering suggested the regulatory roles of long intergenic ncRNAs (lincRNAs) in increasing grafting pepper resistance to Phytophthora capsici

Background As a popular and valuable technique, grafting is widely used to protect against soil-borne diseases and nematodes in vegetable production. Growing evidences have revealed that long intergenic ncRNAs (lincRNAs) are strictly regulated and play essential roles in plants development and stress responses. Nevertheless, genome-wide identification and function deciphering of pepper lincRNAs, especially for their roles in improving grafting pepper resistance to Phytophthora capsici is largely unknown. Results In this study, RNA-seq data of grafting and control pepper plants with or without P. capsici inoculation were used to identify lincRNAs. In total, 2,388 reliable lincRNAs were identified. They were relatively longer and contained few exons than protein-coding genes. Similar to coding genes, lincRNAs had higher densities in euchromatin regions; and longer chromosome transcribed more lincRNAs. Expression pattern profiling suggested that lincRNAs commonly had lower expression than mRNAs. Totally, 607 differentially expressed lincRNAs (DE-lincRANs) were identified, of which 172 were found between P. capsici resistance grafting pepper sample GR and susceptible sample LDS. The neighboring genes of DE-lincRNAs and miRNAs competitively sponged by DE-lincRNAs were identified. Subsequently, the expression level of DE-lincRNAs was further confirmed by qRT-PCR and regulation patterns between DE-lincRNAs and neighboring mRNAs were also validated. Function annotation revealed that DE-lincRNAs increased the resistance of grafting prepper to P. capsici by modulating the expression of disease-defense related genes through cis-regulating and/or lincRNA-miRNA-mRNA interaction networks. Conclusions This study identified pepper lincRNAs and suggested their potential roles in increasing the resistance level of grafting pepper to P. capsici.

Genomic regions and candidate genes linked with Phytophthora capsici root rot resistance in chile pepper (Capsicum annuum L.)

Background Phytophthora root rot, caused by Phytophthora capsici, is a major disease affecting Capsicum production worldwide. A recombinant inbred line (RIL) population derived from the hybridization between ‘Criollo de Morellos-334’ (CM-334), a resistant landrace from Mexico, and ‘Early Jalapeno’, a susceptible cultivar was genotyped using genotyping-by-sequencing (GBS)-derived single nucleotide polymorphism (SNP) markers. A GBS-SNP based genetic linkage map for the RIL population was constructed. Quantitative trait loci (QTL) mapping dissected the genetic architecture of P. capsici resistance and candidate genes linked to resistance for this important disease were identified. Results Development of a genetic linkage map using 1,973 GBS-derived polymorphic SNP markers identified 12 linkage groups corresponding to the 12 chromosomes of chile pepper, with a total length of 1,277.7 cM and a marker density of 1.5 SNP/cM. The maximum gaps between consecutive SNP markers ranged between 1.9 (LG7) and 13.5 cM (LG5). Collinearity between genetic and physical positions of markers reached a maximum of 0.92 for LG8. QTL mapping identified genomic regions associated with P. capsici resistance in chromosomes P5, P8, and P9 that explained between 19.7 and 30.4% of phenotypic variation for resistance. Additive interactions between QTL in chromosomes P5 and P8 were observed. The role of chromosome P5 as major genomic region containing P. capsici resistance QTL was established. Through candidate gene analysis, biological functions associated with response to pathogen infections, regulation of cyclin-dependent protein serine/threonine kinase activity, and epigenetic mechanisms such as DNA methylation were identified. Conclusions Results support the genetic complexity of the P. capsici–Capsicum pathosystem and the possible role of epigenetics in conferring resistance to Phytophthora root rot. Significant genomic regions and candidate genes associated with disease response and gene regulatory activity were identified which allows for a deeper understanding of the genomic landscape of Phytophthora root rot resistance in chile pepper.

A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein trafficking pathway

The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors to enter plant cells and suppress host defense responses. Only few of them are conserved across different strains and species. Such ‘core effectors’ may target hub immunity pathways that are essential during Phytophthora pathogens interacting with their hosts. However, the underlying mechanisms of core RXLRs-mediated host immunity manipulation are largely unknown. Here, we report the functional characterization of a P. capsici RXLR effector, RXLR242. RXLR242 expression is highly induced during the infection process. Its ectopic expression in Nicotiana benthamiana promotes Phytophthora infection. RXLR242 physically interacts with a group of RAB proteins, which belong to the small GTPase family and function in specifying transport pathways in the intracellular membrane trafficking system. RXLR242 impedes the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast by interfering the formation of RABE1-7-labeled vesicles. Further analysis indicated that such phenomenon is resulted from competitive binding of RXLR242 to RABE1-7. RXLR242 also interferes trafficking of the membrane-located receptor FLAGELLIN-SENSING 2 (FLS2) through competitively interacting with RABA4-3. Taken together, our work demonstrates that RXLR242 manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein transporting pathway in plant hosts.