CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita.

BACKGROUND: Plant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately. RESULTS: In the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the ß-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The 'Cas9-free' homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility. CONCLUSION: The present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

A Draft Transcriptome Announcement of Anguina tritici.

Anguina tritici, the wheat seed gall nematode, causes the 'ear-cockle' or seed gall disease of wheat (Triticum sp.), leading to an extensive decline of yield (30-70%) in underdeveloped wheat cultivating countries of the world. The nematode is known to survive in anhydrobiotic conditions for up to 32 years. Here, we present the first transcriptome assembly of A. tritici, which will be a valuable resource for understanding the genes responsible for nematode survival and above-ground plant parasitism. The final 133.2 Mb assembly consists of 105606 open reading frames (including isoforms) with the following BUSCO scores against Nematoda database: 80.3% complete (16.4% single copy and 63.9% duplicated), 2.1% fragmented, and 17.6% missing.

Chemo-profiling of Purpureocillium lilacinum and Paecilomyces variotii isolates using GC-MS analysis, and evaluation of their metabolites against M. incognita.

Nematophagous fungi are the best alternatives to chemical nematicides for managing nematodes considering environmental health. In the current study, activity of metabolites from ten isolates of Purpureocillium lilacinum (Thom) Luangsa-ard (Hypocreales: Ophiocordycipitaceae) and two isolates of Paecilomyces variotii Bainier (Eurotiales: Trichocomaceae), were examined to inhibit the hatching of Meloidogyne incognita (Kofoid & White) Chitwood (Tylenchida: Heteroderidae) eggs. At 100%, 50%, and 25% concentrations, respectively, the culture filtrate of the isolate P. lilacinum 6887 prevented 97.55%, 90.52%, and 62.97% of egg hatching. Out of all the isolates, Pl 6887, Pl 6553, and Pl 2362 showed the greatest results in the hatching inhibition experiment.Gas chromatography-mass spectrometry (GC-MS) analysis revealed a variety of nematicidal compounds from different isolates. A total of seven nematicidal compounds, including four very potent nematicidal fatty acids were found in the isolate Pl 6553. Secondary metabolites of the same isolate possess the highest M. incognita juvenile mortality, i.e., 43.33% and 92% after 48 hrs of treatment at 100 and 200 ppm concentrations, respectively. Significant difference was observed in juvenile mortality percentage among the isolate having highest and lowest nematicidal compounds. Nematicidal fatty acids like myristic and lauric acid were found for the first time in P. lilacinum. Multiple vacuole-like droplets were found inside the unhatched eggs inoculated with the culture filtrate of isolate Pl 6887, and also in the juveniles that perished in the ethyl acetate extract of isolate Pl 6553.

Induced knockdown of Mg-odr-1 and Mg-odr-3 perturbed the host seeking behavior of Meloidogyne graminicola in rice.

Root-knot nematode Meloidogyne graminicola is one of the most destructive plant parasites in upland as well as direct seeded rice. As an integral part of nematode biology, host finding behavior involves perceiving and responding to different chemical cues originating from the rhizosphere. A sustainable management tactic may include retardation of nematode chemoreception that would impair them to detect and discriminate the host stimuli. Deciphering the molecular basis of nematode chemoreception is vital to identify chokepoints for chemical or genetic interventions. However, compared to the well-characterized chemoreception mechanism in model nematode Caenorhabditis elegans, plant nematode chemoreception is yet underexplored. Herein, the full-length cDNA sequences of two chemotaxis-related genes (Mg-odr-1 and Mg-odr-3) were cloned from M. graminicola. Both the genes were markedly upregulated in the early developmental stages of M. graminicola suggesting their involvement in host finding processes. RNAi-induced independent knockdown of Mg-odr-1 and Mg-odr-3 caused behavioral aberration in second-stage juveniles of M. graminicola which in turn perturbed the nematodes' host finding ability and parasitic success inside rice roots. Additionally, nematodes' chemotactic response to different host root exudates, volatile and nonvolatile compounds was affected. Our results demonstrating the role of specific chemosensory genes in modulating M. graminicola host seeking behavior can enrich the existing knowledge of plant nematode chemoreception mechanism, and these genes can be targeted for novel nematicide development or in planta RNAi screens.

Molecular and functional characterization of chemosensory genes from the root-knot nematode Meloidogyne graminicola.

BACKGROUND: Root-knot nematode Meloidogyne graminicola has emerged as a major threat in rice agroecosystems owing to climate change-induced changes in cultivation practices. Synthetic nematicides are continually being withdrawn from the nematode management toolbox because of their ill effects on the environment. A sustainable strategy would be to develop novel nematicides or resistant plants that would target nematode sensory perception, which is a key step in the host finding biology of plant-parasitic nematodes (PPNs). However, compared to the extensive literature on the free-living nematode Caenorhabditis elegans, negligible research has been performed on PPN chemosensory biology. RESULTS: The present study characterizes the five chemosensory genes (Mg-odr-7, Mg-tax-4, Mg-tax-4.1, Mg-osm-9, and Mg-ocr-2) from M. graminicola that are putatively associated with nematode host-finding biology. All the genes were highly transcribed in the early life stages, and RNA interference (RNAi)-induced downregulation of each candidate gene perturbed the normal behavioural phenotypes of M. graminicola, as determined by examining the tracking pattern of juveniles on Pluronic gel medium, attraction to and penetration in rice root tip, and developmental progression in rice root. In addition, a detrimental effect on nematode chemotaxis towards different volatile and nonvolatile organic compounds and host root exudates was documented. CONCLUSION: Our findings enrich the existing literature on PPN chemosensory biology and can supplement future research aimed at identifying a comprehensive chemosensory signal transduction pathway in PPNs.

A nematode-inducible promoter can effectively drives RNAi construct to confer Meloidogyne incognita resistance in tomato.

KEY MESSAGE: Heterologous expression of a nematode-responsive promoter in tomato successfully driven the RNAi constructs to impart root-knot nematode resistance. The root-knot nematode Meloidogyne incognita seriously afflicts the global productivity of tomatoes. Nematode management options are extremely reliant on chemical methods, however, only a handful of nematicides are commercially available. Additionally, nematodes have developed resistance-breaking phenotypes against the commercially available Mi gene-expressing tomatoes. Nematode resistance in crop plants can be enhanced using the bio-safe RNAi technology, in which plants are genetically modified to express nematode gene-specific dsRNA/siRNA molecules. However, the majority of the RNAi crops conferring nematode tolerance have used constitutive promoters, which have many limitations. In the present study, using promoter-GUS fusion, we functionally validated two nematode-inducible root-specific promoters (pAt1g74770 and pAt2g18140, identified from Arabidopsis thaliana) in the Solanum lycopersicum-M. incognita pathosystem. pAt2g18140 was found to be nematode-responsive during 10-21 days post-inoculation (dpi) and became non-responsive during the late infection stage (28 dpi). In contrast, pAt1g74770 remained nematode-responsive for a longer duration (10-28 dpi). Next, a number of transgenic lines were developed that expressed RNAi constructs (independently targeting the M. incognita integrase and splicing factor genes) driven by the pAt1g74770 promoter. M. incognita parasitic success (measured by multiplication factor ratio) in pAt1g74770:integrase and pAt1g74770:splicing factor RNAi lines were significantly reduced by 60.83-74.93% and 69.34-75.31%, respectively, compared to the control. These data were comparable with the RNAi lines having CaMV35S as the promoter. Further, a long-term RNAi effect was evident, because females extracted from transgenic lines were of deformed shape with depleted transcripts of integrase and splicing factor genes. We conclude that pAt1g74770 can be an attractive alternative to drive localized expression of RNAi constructs rather than using a constitutive promoter. The pAt1g74770-driven gene silencing system can be expanded into different plant-nematode interaction models.

Nematode Genome Announcement: A Draft Genome of Seed Gall Nematode, Anguina tritici.

Anguina tritici is the first plant-parasitic nematode described in literature, dating back to the year 1743. It is responsible for causing earcockle (seed gall) and tundu diseases in wheat and rye. Notably, this nematode has been observed to survive in an anhydrobiotic state for up to 32 years within wheat seed galls. These exceptional characteristics have inspired the sequencing of the A. tritici genome. In this study, we present the initial draft genome of A. tritici, obtained using the Illumina MiSeq platform with coverage of 60-fold. The genome is estimated to have a size of 164 Mb and comprises 39,965 protein-coding genes, exhibiting a GC content of 39.1%. The availability of this genome data will serve as a foundation for future functional biological investigations, particularly for genes whose functions remain unknown to this day.

Plant growth promoting Bacillus species elicit defense against Meloidogyne incognita infecting tomato in polyhouse.

The effects of four nematicidal rhizobacterial isolates; Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, and Bacillus cereus on infection and multiplication of root-knot nematode, Meloidogyne incognita on tomato were compared with the application of a chemical nematicide, fluopyram 34.48% SC (Velum Prime). The bio-efficacy trial conducted in pots preinoculated with the above isolates followed by M. incognita inoculation resulted in a significant reduction in percent root galling viz. 91.95 in B. subtilis, 84.21 in B. pumilus, 83.70 in B. megaterium, and 81.8 in B. cereus, at 75 days after inoculation (DAI). The reproduction factor of the nematode was the lowest (15.83) in B. subtilis, followed by B. pumilus (21.00), compared with 48.16 in control, with enhanced photosynthetic and transpiration rates. The mechanism of induced resistance was assessed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for quantification of three key defense genes (PR-1b, JERF3, and CAT) at 0,2,4,8 and16 days DAI. The defence genes, PR-1b, JERF3, and CAT were expressed at 2.5-7.5-folds in rhizobacterialtreated plants, but not in nematicide treatment. The defense enzymes viz., super oxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) when quantified (µmol/mg protein) showed an increase from 1.5 to 17.5 for SOD, 2.1 to 7.8 in PPO, 1.8 to 10.2 in PO, and 1.8 to 8.7 in PAL during 0 to 16 DAI, in rhizobacteria-treated plants.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy.

BACKGROUND: Plant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease. RESULTS: Initially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1-3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, 'transgene-free' T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants. CONCLUSION: Our results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

Gall-specific promoter, an alternative to the constitutive CaMV35S promoter, drives host-derived RNA interference targeting Mi-msp2 gene to confer effective nematode resistance.

One of the major obligate plant parasites causing massive economic crop losses belongs to the class of root-knot nematodes (RKNs). Targeting of major nematode parasitism genes via Host Delivered-RNAi (HD-RNAi) to confer silencing is established as one of the most effective approaches to curb nematode infection. Utilizing nematode-responsive root-specific (NRRS) promoters to design a dsRNA molecule targeting approach to hamper nematode parasitism. Here, a previously validated peroxidase gall specific promoter, pAt2g18140, from Arabidopsis was employed to express the dsRNA construct of the nematode effector gene Mi-msp2 from Meloidogyne incognita. Arabidopsis RNAi lines of CaMV35S::Mi-msp2-RNAi and pAt2g18140::Mi-msp2-RNAi were compared with control plants to assess the decrease in plant nematode infection. When subjected to infection, the maximum reductions in the numbers of galls, females and egg masses in the CaMV35S::Mi-msp2-RNAi lines were 61%, 66% and 95%, respectively, whereas for the pAt2g18140::Mi-msp2-RNAi lines, they were 63%, 68% and 100%, respectively. The reduction in transcript level ranged from 79%-82% for CaMV35S::Mi-msp2-RNAi and 72%-79% for the pAt2g18140::Mi-msp2-RNAi lines. Additionally, a reduction in female size and a subsequent reduction in next-generation fecundity demonstrate the efficacy and potential of the gall specific promoter pAt2g18140 for utilization in the development of HD-RNAi constructs against RKN, as an excellent alternative to the CaMV35S promoter.

Complete Genome Sequence of Brucella abortus 2308, Isolated from an Abortion Storm on a Dairy Farm in India.

The present report communicates the first complete genome sequence of Brucella abortus strain 2308, isolated from an abortion storm on a dairy farm in India. Bacteria were isolated in pure culture from the placentas of aborted calves, and next-generation sequencing (NGS) revealed that the genome sequence length of the isolated strain is 3,285,606 bp, with a GC content of 57.25%, an N50 value of 296,426 bp, and an L50 value of 4, containing 3,119 coding DNA sequences (CDSs), 49 tRNAs, 1 transfer-messenger RNA (tmRNA), and 3 rRNA genes.

Deciphering the mechanism of anhydrobiosis in the entomopathogenic nematode Heterorhabditis indica through comparative transcriptomics.

The entomopathogenic nematode, Heterorhabditis indica, is a popular biocontrol agent of high commercial significance. It possesses tremendous genetic architecture to survive desiccation stress by undergoing anhydrobiosis to increase its lifespan-an attribute exploited in the formulation technology. The comparative transcriptome of unstressed and anhydrobiotic H. indica revealed several previously concealed metabolic events crucial for adapting towards the moisture stress. During the induction of anhydrobiosis in the infective juveniles (IJ), 1584 transcripts were upregulated and 340 downregulated. As a strategy towards anhydrobiotic survival, the IJ showed activation of several genes critical to antioxidant defense, detoxification pathways, signal transduction, unfolded protein response and molecular chaperones and ubiquitin-proteasome system. Differential expression of several genes involved in gluconeogenesis - ß-oxidation of fatty acids, glyoxylate pathway; glyceroneogenesis; fatty acid biosynthesis; amino-acid metabolism - shikimate pathway, sachharopine pathway, kyneurine pathway, lysine biosynthesis; one-carbon metabolism-polyamine pathway, transsulfuration pathway, folate cycle, methionine cycle, nucleotide biosynthesis; mevalonate pathway; and glyceraldehyde-3-phosphate dehydrogenase were also observed. We report the role of shikimate pathway, sachharopine pathway and glyceroneogenesis in anhydrobiotes, and seven classes of repeat proteins, specifically in H. indica for the first time. These results provide insights into anhydrobiotic survival strategies which can be utilized to strengthen the development of novel formulations with enhanced and sustained shelf-life.

Host-delivered RNAi-mediated silencing of the root-knot nematode (Meloidogyne incognita) effector genes, Mi-msp10 and Mi-msp23, confers resistance in Arabidopsis and impairs reproductive ability of the root-knot nematode.

MAIN CONCLUSION: Mi-msp10 and Mi-msp23 effector genes play a significant role during Meloidogyne incognita parasitism on Arabidopsis roots. The role of these genes was confirmed by demonstrating the decrease of the level of susceptibility of Arabidopsis by the  silencing of Mi-msp10 and Mi-msp23 genes using HD-RNAi technology. Root-knot nematodes (RKNs) are the most damaging pathogens severely affecting global food production. The sustainable options to minimize menace of nematode populations through economically feasible measures are limited. Thus, the development of innovative and target-specific strategies that aid in their management is imperative. RNAi technology has emerged as a sustainable and target-specific alternative to control phytonematodes. Here, we characterized two novel subventral gland and dorsal gland-specific effectors, Mi-msp10 and Mi-msp23, to determine their potential effectiveness in controlling M. incognita. Comparative developmental profiling using qRT-PCR revealed higher expression of both effectors in the adult nematode female. Furthermore, functional evaluation of Mi-msp10 and Mi-msp23 dsRNA cassettes was performed using host-delivered RNAi (HD-RNAi) in Arabidopsis. The transgenic lines were examined against M. incognita, and the phenotypic effect of HD-RNAi was evident with a 61% and 51% reduction in gall formation in the Mi-msp10 and Mi-msp23 RNAi lines, respectively. A significant drop in the nematode adult females by 59% for Mi-msp10 and 49% for Mi-msp23-RNAi lines was observed. Similarly, production in egg masses decreased significantly by 76% (Mi-msp10) and 60% (Mi-msp23) for the RNAi lines, which eventually decreased the reproductive factor by 92% and 75%, respectively. The gene expression analysis showed a significant decrease in the transcript level by up to 72% (Mi-msp10) and 66% (Mi-msp23) in M. incognita females feeding on RNAi lines, providing further evidence of effective gene silencing. Overall, our findings provide useful information and support further development of RNAi-based strategies to control M. incognita.

Conferring root-knot nematode resistance via host-delivered RNAi-mediated silencing of four Mi-msp genes in Arabidopsis.

The root-knot nematode (RKN) Meloidogyne incognita is considered one of the most damaging pests among phytonematodes. The majority of nematode oesophageal gland effector genes are indispensable in facilitating M. incognita parasitization of host plants. We report the effect of host-delivered RNAi (HD-RNAi) silencing of four selected M. incognita effector genes, namely, Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24, in Arabidopsis thaliana. Mi-msp5, Mi-msp18 and Mi-msp24, which are dorsal gland genes, were found to be maximally expressed in the adult female stage, whereas Mi-msp3, which is a sub-ventral gland gene, was maximally expressed in an earlier stage. In transgenic plants expressing dsRNA, the reduction in the number of galls on roots was 89 %, 78 %, 86 % and 89 % for the Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24 RNAi events, respectively. Moreover, gene transcript abundance was significantly reduced in RKN females feeding on dsRNA-expressing lines by up to 60 %, 84 %, 31 % and 61 % for Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24, respectively. Furthermore, the M. incognita reproduction factor was reduced up to 71-, 344-, 107- and 114-fold in Arabidopsis plants expressing Mi-msp3, Mi-msp5, Mi-msp18 and Mi-msp24 dsRNA constructs, respectively. This study provides a set of potential target genes to curb nematode infestation in economically important crops via the HD-RNAi approach.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India.

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Development of nematode resistance in Arabidopsis by HD-RNAi-mediated silencing of the effector gene Mi-msp2.

Root-knot nematodes (RKNs) are devastating parasites that infect thousands of plants. As RKN infection is facilitated by oesophageal gland effector genes, one such effector gene, Mi-msp2, was selected for a detailed characterization. Based on domain analysis, the Mi-MSP2 protein contains an ShKT domain, which is likely involved in blocking K+ channels and may help in evading the plant defence response. Expression of the Mi-msp2 gene was higher in juveniles (parasitic stage of RKNs) than in eggs and adults. Stable homozygous transgenic Arabidopsis lines expressing Mi-msp2 dsRNA were generated, and the numbers of galls, females and egg masses were reduced by 52-54%, 60-66% and 84-95%, respectively, in two independent RNAi lines compared with control plants. Furthermore, expression analysis revealed a significant reduction in Mi-msp2 mRNA abundance (up to 88%) in female nematodes feeding on transgenic plants expressing dsRNA, and northern blot analysis confirmed expression of the Mi-msp2 siRNA in the transgenic plants. Interestingly, a significant reduction in the reproduction factor was observed (nearly 40-fold). These data suggest that the Mi-msp2 gene can be used as a potential target for RKN management in crops of economic importance.

Host-mediated RNAi of a Notch-like receptor gene in Meloidogyne incognita induces nematode resistance.

GLP-1 (abnormal germline proliferation) is a Notch-like receptor protein that plays an essential role in pharyngeal development. In this study, an orthologue of Caenorhabditis elegans glp-1 was identified in Meloidogyne incognita. A computational analysis revealed that the orthologue contained almost all the domains present in the C. elegans gene: specifically, the LIN-12/Notch repeat, the ankyrin repeat, a transmembrane domain and different ligand-binding motifs were present in orthologue, but the epidermal growth factor-like motif was not observed. An expression analysis showed differential expression of glp-1 throughout the life cycle of M. incognita, with relatively higher expression in the egg stage. To evaluate the silencing efficacy of Mi-glp-1, transgenic Arabidopsis plants carrying double-stranded RNA constructs of glp-1 were generated, and infection of these plants with M. incognita resulted in a 47-50% reduction in the numbers of galls, females and egg masses. Females obtained from the transgenic RNAi lines exhibited 40-60% reductions in the transcript levels of the targeted glp-1 gene compared with females isolated from the control plants. Second-generation juveniles (J2s), which were descendants of the infected females from the transgenic lines, showed aberrant phenotypes. These J2s exhibited a significant decrease in the overall distance from the stylet to the metacorpus region, and this effect was accompanied by disruption around the metacorporeal bulb of the pharynx. The present study suggests a role for this gene in organ (pharynx) development during embryogenesis in M. incognita and its potential use as a target in the management of nematode infestations in plants.

RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes.

Plant parasitic nematodes cause severe damage and yield loss in major crops all over the world. Available control strategies include use of insecticides/nematicides but these have proved detrimental to the environment, while other strategies like crop rotation and resistant cultivars have serious limitations. This scenario provides an opportunity for the utilization of technological advances like RNA interference (RNAi) to engineer resistance against these devastating parasites. First demonstrated in the model free living nematode, Caenorhabtidis elegans; the phenomenon of RNAi has been successfully used to suppress essential genes of plant parasitic nematodes involved in parasitism, nematode development and mRNA metabolism. Synthetic neurotransmitants mixed with dsRNA solutions are used for in vitro RNAi in plant parasitic nematodes with significant success. However, host delivered in planta RNAi has proved to be a pioneering phenomenon to deliver dsRNAs to feeding nematodes and silence the target genes to achieve resistance. Highly enriched genomic databases are exploited to limit off target effects and ensure sequence specific silencing. Technological advances like gene stacking and use of nematode inducible and tissue specific promoters can further enhance the utility of RNAi based transgenics against plant parasitic nematodes.

Isolation, cloning, and characterization of a cuticle collagen gene, Mi-col-5, in Meloidogyne incognita.

Cuticle collagens form a major part of the nematode cuticle and are responsible for maintaining the overall shape of the animal and its protection from the external environment. Although substantial research on cuticle collagen genes has been carried out in Caenorhabditis elegans, their isolation and characterization in plant parasitic nematodes have been limited to a few genes only. In this study, a cuticle collagen gene, Mi-col-5, was isolated from root-knot nematode, Meloidogyne incognita. A partial segment of 402 bp was first cloned and analyzed on Gbrowse followed by subsequent cloning of the 1047 bp long full cDNA specifying the open reading frame. The deduced amino acid sequence showed 92% sequence identity with that of Mj-col-5. However, a transmembrane helix was predicted in Mi-col-5 which was not present in Mj-col-5. The conserved pattern of cysteine residues in Mi-col-5 suggested that it belonged to group 2 of nematode cuticle collagens but with a longer carboxy terminal region as was the case with Mj-col-5. Domain prediction revealed the presence of a nematode cuticle collagen N terminal domain and a pfam collagen domain along with collagen triple helix repeats. A phylogenetic tree based on the amino acid sequences showed evolutionary relationship of Mi-col-5 with cuticle collagens genes of other nematodes. 3D models for Mi-col-5 were predicted with the best confidence score of -2.78. Expression of Mi-col-5 transcript was found to be maximum in egg masses followed by adult females and J2s suggesting its role in the early stages of the development of the nematode during its life cycle.

Identification, Validation and Utilization of Novel Nematode-Responsive Root-Specific Promoters in Arabidopsis for Inducing Host-Delivered RNAi Mediated Root-Knot Nematode Resistance.

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants.