Comparative methylome reveals regulatory roles of DNA methylation in melon resistance to Podosphaera xanthii.

Powdery mildew caused by Podosphaera xanthii (P. xanthii) severely endangers melon (Cucumis melo L.) production, while the mechanistic understanding about its resistance to powdery mildew remains largely limited. In this study, we integrated transcriptomic and methylomic analyses to explore whether DNA methylation was involved in modulating transcriptional acclimation of melon to P. xanthii infection. Net photosynthetic rate (Pn), stomatal conductance (Gs), actual photochemical efficiency (ФPSII) and maximum PSII quantum yield (Fv/Fm) were significantly decreased in P. xanthii-infected plants relative to uninfected ones (Control), revealing apparent physiological disorders. Totally 4808 differentially expressed genes (DEGs) were identified by global analysis of gene expression in Control and P. xanthii-infected plants. Comparative methylome uncovered that 932 DEGs were associated with hypermethylation, while 603 DEGs were associated with hypomethylation in melon upon P. xanthii infection. Among these differential methylation-involved DEGs, a set of resistance-related genes including R genes and candidate genes in metabolic and defense pathways were further identified, demonstrating that DNA methylation might function as a new regulatory layer for melon resistance to P. xanthii infection. Altogether our study sheds new insights into the molecular mechanisms of melon against powdery mildew and provides some potential targets for improving melon disease resistance in future.

A single gene in Fusarium oxysporum limits host range.

Fusarium oxysoporum f. sp. radicis-cucumerinum (Forc) is able to cause disease in cucumber, melon, and watermelon, while F. oxysporum f. sp. melonis (Fom) can only infect melon plants. Earlier research showed that mobile chromosomes in Forc and Fom determine the difference in host range between Forc and Fom. By closely comparing these pathogenicity chromosomes combined with RNA-sequencing data, we selected 11 candidate genes that we tested for involvement in the difference in host range between Forc and Fom. One of these candidates is a putative effector gene on the Fom pathogenicity chromosome that has nonidentical homologs on the Forc pathogenicity chromosome. Four independent Forc transformants with this gene from Fom showed strongly reduced or no pathogenicity towards cucumber, while retaining pathogenicity towards melon and watermelon. This suggests that the protein encoded by this gene is recognized by an immune receptor in cucumber plants. This is the first time that a single gene has been demonstrated to determine a difference in host specificity between formae speciales of F. oxysporum.

The Functional Characterization of Podosphaera xanthii Candidate Effector Genes Reveals Novel Target Functions for Fungal Pathogenicity.

Podosphaera xanthii is the main causal agent of powdery mildew disease in cucurbits. In a previous study, we determined that P. xanthii expresses approximately 50 Podosphaera effector candidates (PECs), identified based on the presence of a predicted signal peptide and the absence of functional annotation. In this work, we used host-induced gene silencing (HIGS), employing Agrobacterium tumefaciens as a vector for the delivery of the silencing constructs (ATM-HIGS), to identify genes involved in early plant-pathogen interaction. The analysis of seven selected PEC-encoding genes showed that six of them, PEC007, PEC009, PEC019, PEC032, PEC034, and PEC054, are required for P. xanthii pathogenesis, as revealed by reduced fungal growth and increased production of hydrogen peroxide by host cells. In addition, protein models and protein-ligand predictions allowed us to identify putative functions for these candidates. The biochemical activities of PEC019, PEC032, and PEC054 were elucidated using their corresponding proteins expressed in Escherichia coli. These proteins were confirmed as phospholipid-binding protein, α-mannosidase, and cellulose-binding protein. Further, BLAST searches showed that these three effectors are widely distributed in phytopathogenic fungi. These results suggest novel targets for fungal effectors, such as host-cell plasma membrane, host-cell glycosylation, and damage-associated molecular pattern-triggered immunity.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Characterization of CNL like protein fragment (CNL-LPF) from mature Lageneria siceraria seeds.

Coiled coil domain-nucleotide binding site-leucine rich repeat (CC-NBS-LRR; CNL) proteins are highly conserved family of plant disease resistance proteins, remarkably comprise of coiled-coil domain, which plays significant role in plant innate immunity. The present study reports that moderately elicited oligomerization of plant CNL like protein fragment (CNL-LPF) in presence of ATP/Mg using various biophysical methods Circular dichroism (CD) results depicted a substantial increase in ß-sheet structure content of CNL-LPF. ATP/Mg induced conformational change in protein was observed by increase in blue shift with extrinsic fluorescence measurement, which indicates the exposure of hydrophobic regions of CNL-LPF and leads to self-association i.e. oligomerization. Likewise, cluster of protein oligomer and alteration in protein surface morphology were observed in presence of ATP/Mg by Transmission electron microscopy (TEM) and Atomic force microscopy (AFM), respectively. Also, augmented antiproliferation of HT1376 cells (urinary bladder cancer cell lines) was observed by CNL-LPF in presence of ATP/Mg. In conclusion, the current study illustrates that extent of CNL-LPF oligomerization was enhanced in presence of ATP/Mg (as compared to its absence). Utilization of enhanced oligomerization property of CNL-LPF as an anti-proliferative agent needs more assessment.

Polyclonal Antibody Development Against Purified CC-NBS-LRR like Protein Fragment from Mature Lageneria siceraria Seeds and Immunolocalization.

CC-NBS-LRR (CNL) plant proteins are related with highly conserved family of disease resistance protein distinguished by a coiled-coil domain, which plays an important role in innate immunity. The present study reports the purification and identification of CNL like protein fragment (CNL-LPF) by two step chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/MS), respectively. Furthermore, current study also illustrated the development of polyclonal antibody against purified CNL-LPF, which was used for immunolocalization of CNL-LPF in cytoplasm of cotyledon, using Fluorescence microscopy and Transmission electron microscopy. Lastly, present study also demonstrates in vitro oligomerization of purified CNL-LPF with multiple bands on 4-10 % gradient native-PAGE; each band representing a small fraction of each oligomer population as evident by immunoblots. In conclusion, the current study deals with the purification and polyclonal antibody development against CNL-LPF.

The latex sap of the 'Old World Plant' Lagenaria siceraria with potent lectin activity mitigates neoplastic malignancy targeting neovasculature and cell death.

Lifestyle and dietary modifications have contributed much to somatic genetic alteration which has concomitantly led to increase in malignant diseases. Henceforth, plant based and dietary interventions to mitigate and impede oncogenic transformation are in great demand. We investigated the latex sap (LSL) of the dietary Lagenaria siceraria vegetable, the first domesticated plant species with the potent lectin activity for its functional role against the tumor progression and its mechanism. LSL has markedly stimulated proliferation of lymphocytes and displayed strong cytotoxic activity against cancer both in-vitro and in-vivo. The tumor regression was paralleled with drastic reduction in tumoral neovasculature as evidenced from angiogenic parameters and abrogated related gene expressions. LSL has also triggered apoptotic signaling cascade in cancer cells through activation of caspase-3 mediated activation of endonuclease and inducing apoptotic cellular events. Collectively our study provides tangible evidences that latex sap from L. siceraria with immunopotentiating ability significantly regresses the tumor progression by targeting angiogenesis and inducing cell death.

Greater efficacy of the ECMS-oil adjuvant over other formulations on immune responses against Bordetella bronchiseptica in rabbits and the underlying mechanism.

In this study, the adjuvant effects of the extract of Cochinchina momordica seed ECMS+oil, oil alone, ECMS alone, conventional alum adjuvant on inactivated Bordetella bronchiseptica (Bb) vaccine or control using antigen alone without adjuvant were evaluated along with the underlying mechanism. The results in experiment A demonstrated that antibody levels in Bb whole cell protein in the ECMS800µg+oil group were significantly higher than in the other adjuvant groups (p<0.05) on day 21. The agglutination antibody titer was also higher than the other groups (p<0.05) on day 37. The ECMS800µg+oil group improved cellular immune responses compared to other adjuvant groups, including control using antigen alone without adjuvant and the PBS group (p<0.05). After Bb challenge, the ECMS800µg+oil group showed the highest protection rate, which was significantly higher than ECMS alone or control using antigen alone without adjuvant and the PBS group (p<0.05 and p<0.01). IgA cells in the ECMS800µg+oil group differed significantly from the IgA cells of other groups in the lungs (p<0.01). The results of cell recruitment showed that the number of lymphocytes in the ECMS400µg+oil were higher than the number of cells for other groups except the ECMS(100µg/800µg)+oil groups (p<0.05). Intermediate cells in the ECMS(100µg/400µg)+oil groups were higher than the number of cells for other groups, including the control using antigen alone group (p<0.05). Neutrophils in the ECMS(100µg/400µg/800µg)+oil groups were significantly higher than the ECMS 800µg and control using antigen alone groups (p<0.05). White blood cells in the ECMS100µg+oil group were significantly higher than the oil, ECMS800µg and control using antigen alone groups (p<0.05). IL-2 expression in the ECMS800µg+oil group was significantly higher than other groups, except for the ECMS400µg+oil group (p<0.05). IL-4 expression in the ECMS800µg+oil group was significantly higher than other groups (p<0.05). GATA3 in the ECMS800µg+oil groups was significantly higher than the oil, ECMS800µg and control using antigen alone group (p<0.05). ECMS-oil adjuvant mixture could most effectively protect B. bronchiseptica immunized rabbits and, therefore, could be an alternative way of improving B. bronchiseptica vaccination in rabbits.

Root exudates from grafted-root watermelon showed a certain contribution in inhibiting Fusarium oxysporum f. sp. niveum.

Grafting watermelon onto bottle gourd rootstock is commonly used method to generate resistance to Fusarium oxysporum f. sp. niveum (FON), but knowledge of the effect of the root exudates of grafted watermelon on this soil-borne pathogen in rhizosphere remains limited. To investigate the root exudate profiles of the own-root bottle gourd, grafted-root watermelon and own-root watermelon, recirculating hydroponic culture system was developed to continuously trap these root exudates. Both conidial germination and growth of FON were significantly decreased in the presence of root exudates from the grafted-root watermelon compared with the own-root watermelon. HPLC analysis revealed that the composition of the root exudates released by the grafted-root watermelon differed not only from the own-root watermelon but also from the bottle gourd rootstock plants. We identified salicylic acid in all 3 root exudates, chlorogenic acid and caffeic acid in root exudates from own-root bottle gourd and grafted-root watermelon but not own-root watermelon, and abundant cinnamic acid only in own-root watermelon root exudates. The chlorogenic and caffeic acid were candidates for potentiating the enhanced resistance of the grafted watermelon to FON, therefore we tested the effects of the two compounds on the conidial germination and growth of FON. Both phenolic acids inhibited FON conidial germination and growth in a dose-dependent manner, and FON was much more susceptible to chlorogenic acid than to caffeic acid. In conclusion, the key factor in attaining the resistance to Fusarium wilt is grafting on the non-host root stock, however, the root exudates profile also showed some contribution in inhibiting FON. These results will help to better clarify the disease resistance mechanisms of grafted-root watermelon based on plant-microbe communication and will guide the improvement of strategies against Fusarium-mediated wilt of watermelon plants.

Frequent loss of lineages and deficient duplications accounted for low copy number of disease resistance genes in Cucurbitaceae.

BACKGROUND: The sequenced genomes of cucumber, melon and watermelon have relatively few R-genes, with 70, 75 and 55 copies only, respectively. The mechanism for low copy number of R-genes in Cucurbitaceae genomes remains unknown. RESULTS: Manual annotation of R-genes in the sequenced genomes of Cucurbitaceae species showed that approximately half of them are pseudogenes. Comparative analysis of R-genes showed frequent loss of R-gene loci in different Cucurbitaceae species. Phylogenetic analysis, data mining and PCR cloning using degenerate primers indicated that Cucurbitaceae has limited number of R-gene lineages (subfamilies). Comparison between R-genes from Cucurbitaceae and those from poplar and soybean suggested frequent loss of R-gene lineages in Cucurbitaceae. Furthermore, the average number of R-genes per lineage in Cucurbitaceae species is approximately 1/3 that in soybean or poplar. Therefore, both loss of lineages and deficient duplications in extant lineages accounted for the low copy number of R-genes in Cucurbitaceae. No extensive chimeras of R-genes were found in any of the sequenced Cucurbitaceae genomes. Nevertheless, one lineage of R-genes from Trichosanthes kirilowii, a wild Cucurbitaceae species, exhibits chimeric structures caused by gene conversions, and may contain a large number of distinct R-genes in natural populations. CONCLUSIONS: Cucurbitaceae species have limited number of R-gene lineages and each genome harbors relatively few R-genes. The scarcity of R-genes in Cucurbitaceae species was due to frequent loss of R-gene lineages and infrequent duplications in extant lineages. The evolutionary mechanisms for large variation of copy number of R-genes in different plant species were discussed.

A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes.

A reliable and species-specific real-time quantitative polymerase chain reaction (qPCR) assay was developed for detection of the complex soilborne anamorphic fungus Fusarium oxysporum. The new primer pair, designed on the translation elongation factor 1-α gene with an amplicon of 142 bp, was highly specific to F. oxysporum without cross reactions with other Fusarium spp. The protocol was applied to grafted melon plants for the detection and quantification of F. oxysporum f. sp. melonis, a devastating pathogen of this cucurbit. Grafting technologies are widely used in melon to confer resistance against new virulent races of F. oxysporum f. sp. melonis, while maintaining the properties of valuable commercial varieties. However, the effects on the vascular pathogen colonization have not been fully investigated. Analyses were performed on 'Charentais-T' (susceptible) and 'Nad-1' (resistant) melon cultivars, both used either as rootstock and scion, and inoculated with F. oxysporum f. sp. melonis race 1 and race 1,2. Pathogen development was compared using qPCR and isolations from stem tissues. Early asymptomatic melon infections were detected with a quantification limit of 1 pg of fungal DNA. The qPCR protocol clearly showed that fungal development was highly affected by host-pathogen interaction (compatible or incompatible) and time (days postinoculation). The principal significant effect (P ≤ 0.01) on fungal development was due to the melon genotype used as rootstock, and this effect had a significant interaction with time and F. oxysporum f. sp. melonis race. In particular, the amount of race 1,2 DNA was significantly higher compared with that estimated for race 1 in the incompatible interaction at 18 days postinoculation. The two fungal races were always present in both the rootstock and scion of grafted plants in either the compatible or incompatible interaction.

The antagonistic strain Bacillus subtilis†UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses.

Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.

A mutant of the major melon allergen, Cuc m 2, with reduced IgE binding capacity is a good candidate for specific immunotherapy.

Hypoallergenic mutants with reduced IgE-binding capacity but which show a similar T-cell response to the corresponding natural allergen are ideal tools for immunotherapy, for preventing a possible anaphylactic shock. An IgE conformational epitope has been identified in Cuc m 2, the major allergen and profilin from melon. Since this epitope is highly conserved in most pollen profilins, it may contribute to an explanation of cross-reactivity between pollen and food profilins. Mutants (Mut 1 and Mut 2) were generated by changing specific residues of the Cuc m 2 epitope to alanine, produced in Escherichia coli, and purified by chromatographic methods. Mut 1 showed a slight reduction in IgE binding but an allergenic activity that was similar to recombinant Cuc m 2, as measured by basophil activation test (BAT) and skin prick test (SPT). By contrast, Mut 2 displayed a substantial reduction in IgE-binding capacity (57%) and positive responses, as determined by BAT (33%) and SPT (50%), when compared to those of rCuc m 2. However, the T-cell proliferation and cytokine production induced by Mut 2 and rCuc m 2 were similar. Thus, this mutant represent potential candidate for immunotherapy of profilin allergies.

The nature of melon allergy in ragweed-allergic subjects: A study of 1000 patients.

Previous studies suggest cross-reactivity between specific ragweed pollen and melon allergens. This study was designed to clarify the origin of the cross-reactivity between ragweed pollen and the gourd family. One thousand ragweed-allergic subjects were interviewed about the presence of oral allergy syndrome (OAS) induced by melon or watermelon and were divided into reactive to ≤3 seasonal allergen sources or >3 seasonal allergen sources. Patients reporting melon and/or watermelon allergy underwent a skin-prick test (SPT) with fresh melon and, after 2006, also with profilin-enriched date palm pollen extract. Because no IgE reactivity to melon extract was detected in vitro, ELISA was performed using date palm pollen extract, and inhibition experiments were performed using grass pollen, date palm profilin, and bovine serum albumin (BSA) as inhibitors. Six hundred forty-six and 354 subjects reacted to ≤3 seasonal allergens or >3 seasonal allergens, respectively; 4/646 (1%) and 81/354 (23%) reported a history of melon/watermelon-induced OAS (p < 0.0001). Forty-three of 46 (93%) melon reactors scored positive on SPT with the profilin-enriched extract, which was positive in 0/2 (0%) versus 43/44 (98%) reactive to ≤3 or >3 seasonal allergen sources, respectively (p < 0.0001). in vitro, serum from melon-allergic subjects showed a strong IgE reactivity to the profilin-enriched date palm pollen extract, which was abolished by preabsorption with both grass pollen extract and date palm pollen extract, but not by BSA. In ragweed pollen-allergic subjects, melon allergy is most likely associated with cross-sensitization to the plant pan-allergen profilin and not to specific ragweed pollen allergens. This study confirms the association between profilin sensitization and melon allergy.

Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.