Deciphering the Role of Mucosal Immune Responses and the Cervicovaginal Microbiome in Resistance to HIV Infection in HIV-Exposed Seronegative (HESN) Women.

The female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection. IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.

Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants.

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species.

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns.

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.

Delineation of Genotype-by-Environment interactions for identification and validation of resistant genotypes in mungbean to root-knot nematode (Meloidogyne incognita) using GGE biplot.

Susceptibility to root-knot nematodes (Meloidogyne spp.) is one of the major factors limiting mungbean production in South and South-East Asia. Host-pest-environment interaction in mungbean and root-knot nematode (M. incognita) was investigated in multi-location field evaluation using 38 promising mungbean genotypes extracted from initial evaluation of 250 genotypes under sick plots considering second stage freshly hatched juvenile as inoculants. The extent of environmental and genotype-by-environment interactions (GGE) was assessed to comprehend the dynamism of resistance and identification of durable resistant mungbean genotypes. Among environmental factors, nematode activity was highly influenced by rainfall and minimum temperature. The GGE biplot and multiple comparison tests detected a higher proportion of genotype × environment (GE) interaction followed by genotype and environment on number of nematode galls, gall index and reproduction factor. The first two principal components (PCs) explained 64.33% and 66.99% of the total variation of the environment-centered gall scoring and reproduction factor data, respectively. The high GE variation indicated the presence of non-cross over interactions which justify the necessities of multi-location testing. Detection of non-redundant testing locations would expedite optimum resource utilization in future. The GGE biplot analysis identified genotypes such as PM-10-12, IPM-410-3 and NVL-641 as the outperforming and desirable genotypes with durable resistance against M. incognita which can be exploited in mungbean breeding programmes globally. On the contrary, the highest gall scoring and reproduction factor were recorded in genotype IPM-9901-8. Computation of confidence interval (CI) at 95% level through bootstrapping increased precision of GGE biplot towards genotype recommendation. Furthermore, total phenol content, ascorbic acid, phenlylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) activities were also higher in identified resistant genotypes and this information would be useful for devising mungbean breeding strategies in future for resistance against root-knot nematodes.

Construction of a high density linkage map and genome dissection of bruchid resistance in zombi pea (Vigna vexillata (L.) A. Rich).

Zombi pea (Vigna vexillata) is a legume crop that is resistant to several biotic and abiotic stresses. Callosobruchus maculatus and Callosobruchus chinensis are serious stored-insect pests of legume crops. We constructed a high-density linkage map and performed quantitative trait loci (QTLs) mapping for resistance to these insect species in zombi pea. An F2 population of 198 individuals from a cross between 'TVNu 240' (resistant) and 'TVNu 1623' (susceptible) varieties was used to construct a linkage map of 6,529 single nucleotide polymorphism markers generated from sequencing amplified fragments of specific loci. The map comprised 11 linkage groups, spanning 1,740.9 cM, with an average of 593.5 markers per linkage group and an average distance of 0.27 cM between markers. High levels of micro-synteny between V. vexillata and cowpea (Vigna unguiculata), mungbean (Vigna radiata), azuki bean (Vigna angularis) and common bean (Phaseolus vulgaris) were found. One major and three minor QTLs for C. chinensis resistance and one major and one minor QTLs for C. maculatus resistance were identified. The major QTLs for resistance to C. chinensis and C. maculatus appeared to be the same locus. The linkage map developed in this study will facilitate the identification of useful genes/QTLs in zombi pea.

Complex molecular mechanisms underlying MYMIV-resistance in Vigna mungo revealed by comparative transcriptome profiling.

Mungbean Yellow Mosaic India Virus (MYMIV)-infection creates major hindrance in V. mungo cultivation and poses significant threat to other grain legume production. Symptoms associated include severe patho-physiological alterations characterized by chlorotic foliar lesion accompanied by reduced growth. However, dissection of the host's defense machinery remains a tough challenge due to limited of host's genomic resources. A comparative RNA-Seq transcriptomes of resistant (VM84) and susceptible (T9) plants was carried out to identify genes potentially involved in V. mungo resistance against MYMIV. Distinct gene expression landscapes were observed in VM84 and T9 with 2158 and 1679 differentially expressed genes (DEGs), respectively. Transcriptomic responses in VM84 reflect a prompt and intense immune reaction demonstrating an efficient pathogen surveillance leading to activation of basal and induced immune responses. Functional analysis of the altered DEGs identified multiple regulatory pathways to be activated or repressed over time. Up-regulation of DEGs including NB-LRR, WRKY33, ankyrin, argonaute and NAC transcription factor revealed an insight on their potential roles in MYMIV-resistance; and qPCR validation shows a propensity of their accumulation in VM84. Analyses of the current RNA-Seq dataset contribute immensely to decipher molecular responses that underlie MYMIV-resistance and will aid in the improvement strategy of V. mungo and other legumes through comparative functional genomics.

Exogenous application of methyl jasmonate induces defense response and develops tolerance against mungbean yellow mosaic India virus in Vigna mungo.

Vigna mungo (L.)Hepper is an economically important leguminous crop in south-east Asia. However, its production is severely affected by Mungbean yellow mosaic India virus (MYMIV). It is well established that methyl jasmonate (MeJA) is effective in inducing resistance against pathogens in several plants. To assess the role of MeJA in developing MYMIV tolerance in V. mungo, we analysed time-dependent biochemical and molecular responses of MYMIV susceptible V. mungo after exogenous application of different MeJA concentrations, followed by MYMIV infection. Our analysis revealed that exogenous application of different concentrations of MeJA resulted in decreased levels of malondialdehyde with higher membrane stability index values in MYMIV susceptible V. mungo, suggesting the protective role of MeJA through restoring the membrane stability. Moreover, the level of expression of different antioxidative enzymes revealed that exogenous MeJA is also very effective in ROS homeostasis maintenance. Enhanced expressions of the defence marker genes lipoxygenase and phenylalanine ammonia-lyase and the reduced expression of the MYMIV coat-protein encoding gene in all MeJA treated plants post MYMIV infection revealed that exogenous application of MeJA is effective for MYMIV tolerance in V. mungo. Our findings provide new insights into the physiological and molecular mechanisms of MYMIV tolerance in Vigna induced by MeJA.

Diallel crosses for resistance to Macrophomina phaseolina and Thanatephorus cucumeris on cowpea.

This study aimed at identifying and selecting through partial diallel analysis, segregating populations of cowpea resistant to Macrophomina phaseolina and Thanatephorus cucumeris, based on the evaluation of general (GCA) and specific combining ability (SCA), involved in the genetic control of resistance. For this reason, 19 grouped cowpea genotypes, considering the resistance to these pathogens, were crossed in partial diallel scheme 14 x 5, during 2013 and 2014. The 70 F2 populations and the 19 parents were evaluated in a greenhouse as the reaction to pathogens, separately. The diallel analysis was performed according to the model of partial diallel proposed by Geraldi and Miranda Filho (1988). The additive effects predominated in the genetic control of the traits severity of charcoal rot (SEV) and area under the disease progress curve (AUDPC) to web blight, enabling the achievement of genetic gain with selection of resistant strains. Analyzing the effects of GCA, the parents BR 14-Mulato, BRS Tumucumaque and BRS Guariba, have a higher concentration of favorable alleles, highlighting, according to the values of SCA, the combinations BR 14-Mulato x MNC02-675F-4-10, BRS Tumucumaque x IT98K-1092-1, BRS Tumucumaque x MNC02-675F-4-10, BRS Tumucumaque x MNC02-675F-9-2, BRS Guariba x IT98K-1092-1, BRS Guariba x MNC02-675F-4-9, and BRS Guariba x MNC02-675F-4-10, as the most promising and indicated to obtain lines resistant to M. phaseolina and T. cucumeris in cowpea, simultaneously.

Molecular and biochemical characterization of a Vigna mungo MAP kinase associated with Mungbean Yellow Mosaic India Virus infection and deciphering its role in restricting the virus multiplication.

Yellow Mosaic Disease caused by the begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) severely affects many economically important legumes. Recent investigations in Vigna mungo - MYMIV incompatible interaction identified a MAPK homolog in the defense signaling pathway. An important branch of immunity involves phosphorylation by evolutionary conserved Mitogen-activated protein kinases (MAPK) that transduce signals of pathogen invasion to downstream molecules leading to diverse immune responses. However, most of the knowledge of MAPKs is derived from model crops, and functions of these versatile kinases are little explored in legumes. Here we report characterization of a MAP kinase (VmMAPK1), which was induced upon MYMIV-inoculation in resistant V. mungo. Phylogenetic analysis revealed that VmMAPK1 is closely related to other plant-stress-responsive MAPKs. Both mRNA and protein of VmMAPK1 were accumulated upon MYMIV infection. The VmMAPK1 protein localized in the nucleus as well as cytoplasm and possessed phosphorylation activity in vitro. A detailed biochemical characterization of purified recombinant VmMAPK1 demonstrated an intramolecular mechanism of autophosphorylation and self-catalyzed phosphate incorporation on both threonine and tyrosine residues. The Vmax and Km values of recombinant VmMAPK1 for ATP were 6.292nmol/mg/min and 0.7978µM, respectively. Furthermore, the ability of VmMAPK1 to restrict MYMIV multiplication was validated by its ectopic expression in transgenic tobacco. Importantly, overexpression of VmMAPK1 resulted in the considerable upregulation of defense-responsive marker PR genes. Thus, the present data suggests the critical role of VmMAPK1 in suppressing MYMIV multiplication presumably through SA-mediated signaling pathway and inducing PR genes establishing the significant implications in understanding MAP kinase gene function during Vigna-MYMIV interaction; and hence paves the way for introgression of resistance in leguminous crops susceptible to MYMIV.

Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV).

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. BIOLOGICAL SIGNIFICANCE: This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV.

Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene.

Identification of single nucleotide polymorphism markers associated with resistance to bruchids (Callosobruchus spp.) in wild mungbean (Vigna radiata var. sublobata) and cultivated V. radiata through genotyping by sequencing and quantitative trait locus analysis.

BACKGROUND: Bruchid beetles are an important storage pest of grain legumes. Callosobruchus sp. infect mungbean (Vigna radiata) at low levels in the field, multiply during grain storage and can destroy seed stocks in a few months. Resistance against bruchid beetles has been found in wild mungbean V. radiata var. sublobata TC1966 and in cultivated mungbean line V2802. RESULTS: Bruchid resistance data were obtained from recombinant inbred line populations TC1966 (V. radiata var. sublobata) × NM92 (F12) and V2802 (V. radiata) × NM94 (F7). More than 6,000 single nucleotide polymorphic markers were generated through genotyping by sequencing (GBS) for each of these populations and were used to map bruchid resistance genes. One highly significant quantitative trait locus (QTL) associated with bruchid resistance was mapped to chromosome 5 on genetic maps of both populations, suggesting that TC1966 and V2802 contain the same resistance locus. Co-segregation of all markers associated with resistance indicated the presence of only one major resistance QTL on chromosome 5, while QTL analysis based on physical map positions of the markers suggested the presence of multiple QTLs on different chromosomes. The diagnostic capacity of the identified molecular markers located in the QTL to correctly predict resistance was up to 100 %. CONCLUSIONS: Molecular markers tightly linked to bruchid resistance loci of two different mungbean resistance sources were developed and validated. These markers are highly useful for developing resistant lines.