Tushar K. Dutta

The status of RNAi-based transgenic research in plant nematology

With the understanding of nematode-plant interactions at the molecular level, new avenues for engineering resistance have opened up, with RNA interference being one of them. Induction of RNAi by delivering double-stranded RNA (dsRNA) has been very successful in the model non-parasitic nematode, Caenorhabditis elegans, while in plant nematodes, dsRNA delivery has been accomplished by soaking nematodes with dsRNA solution mixed with synthetic neurostimulants. The success of in vitro RNAi of target genes has inspired the use of in planta delivery of dsRNA to feeding nematodes. The most convincing success of host-delivered RNAi has been achieved against root-knot nematodes. Plant-mediated RNAi has been shown to lead to the specific down-regulation of target genes in invading nematodes, which had a profound effect on nematode development. RNAi-based transgenics are advantageous as they do not produce any functional foreign proteins and target organisms in a sequence-specific manner. Although the development of RNAi-based transgenics against plant nematodes is still in the preliminary stage, they offer novel management strategy for the future.

Comparison of host recognition, invasion, development and reproduction of Meloidogyne graminicola and M. incognita on rice and tomato

The rice root-knot nematode Meloidogyne graminicola normally infects rice, wheat and several other graminaceous plants. Meloidogyne incognita is a serious pest of dicotyledonous crops, although it can infect and reproduce on some cereals. This paper demonstrates and compares host recognition, development and reproduction of these two species of root-knot nematodes on rice and tomato plants. Attraction bioassays in pluronic gel clearly showed that M. incognita preferred tomato roots to rice or mustard roots, whilst M. graminicola was more attracted towards rice compared with tomato or mustard roots. Based on the attraction data from this study, it can be hypothesised that either: i ) the blend of attractants and repellents are different in good and poor hosts; or ii ) relatively long-range attractants, together with shorter-range repellents, might affect nematode movement patterns. Some host specific attractants might also be involved. Meloidogyne incognita was able to invade and develop to adult female but did not produce eggs in rice roots. By contrast, M. graminicola developed and reproduced faster on both rice and tomato plants compared with M. incognita. Nevertheless, second-stage juveniles of both these root-knot nematodes showed a similar pattern of distribution inside the roots, preferring to accumulate at the root tips of rice or in the vascular cylinder and cortical region of tomato.

Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes.

Root-knot nematodes (Meloidogyne incognita) cause substantial yield losses in vegetables worldwide, and are difficult to manage. Continuous withdrawal of environmentally-harmful nematicides from the global market warrants the need for novel nematode management strategies. Utility of host-delivered RNAi has been demonstrated in several plants (Arabidopsis, tobacco, and soybean) that exhibited resistance against root-knot and cyst nematodes. Herein, a M. incognita-specific protease gene, cathepsin L cysteine proteinase (Mi-cpl-1), was targeted to generate tomato transgenic lines to evaluate the genetically modified nematode resistance. In vitro knockdown of Mi-cpl-1 gene led to the reduced attraction and penetration of M. incognita in tomato, suggesting the involvement of Mi-cpl-1 in nematode parasitism. Transgenic expression of the RNAi construct of Mi-cpl-1 gene resulted in 60–80% reduction in infection and multiplication of M. incognita in tomato. Evidence for in vitro and in vivo silencing of Mi-cpl-1 was confirmed by expression analysis using quantitative PCR. Our study demonstrates that Mi-cpl-1 plays crucial role during plant-nematode interaction and plant-mediated downregulation of this gene elicits detrimental effect on M. incognita development, reinforcing the potential of RNAi technology for management of phytonematodes in crop plants.

Comparing the defence-related gene expression changes upon root-knot nematode attack in susceptible versus resistant cultivars of rice.

Rice is one of the major staple food crops in the world and an excellent model system for studying monocotyledonous plants. Diseases caused by nematodes in rice are well documented and among them, root-knot nematode (RKN), Meloidogyne graminicola, causes extensive yield decline. It is therefore necessary to identify novel sources of natural resistance to RKN in rice and to investigate the rice-RKN interaction in detail to understand the basal plant defence mechanisms and nematode manipulation of the host physiology. To this end, six different cultivars of rice were initially screened for RKN infection and development; Pusa 1121 and Vandana were found to be most susceptible and resistant to RKN infection, respectively. In order to investigate the role of major hormone-regulated plant defence pathways in compatible/incompatible rice-RKN interaction, some well-identified marker genes involved in salicylate/jasmonate/ethylene pathway were evaluated for their differential expression through qRT-PCR. In general, our study shows a remarkable discrepancy in the expression pattern of those genes between compatible and incompatible rice-RKN interaction. As most information on the molecular interplay between plants and nematodes were generated on dicotyledonous plants, the current study will strengthen our basic understanding of plant-nematode interaction in the monocot crops, which will aid in defining future strategies for best plant health measures.

Expression of a Cystatin Transgene in Eggplant Provides Resistance to Root-knot Nematode, Meloidogyne incognita

Root-knot nematodes (RKN) cause substantial yield decline in eggplant and sustainable management options to minimize crop damage due to nematodes are still limited. A number of genetic engineering strategies have been developed to disrupt the successful plant–nematode interactions. Among them, delivery of proteinase inhibitors from the plant to perturb nematode development and reproduction is arguably the most effective strategy. In the present study, transgenic eggplant expressing a modified rice cystatin (OC-IΔD86) gene under the control of the root-specific promoter, TUB-1, was generated to evaluate the genetically modified nematode resistance. Five putative transformants were selected through PCR and genomic Southern blot analysis. Expression of the cystatin transgene was confirmed in all the events using western blotting, ELISA and qPCR assay. Upon challenge inoculation, all the transgenic events exhibited a detrimental effect on RKN development and reproduction. The best transgenic line (a single copy event) showed 78.3% inhibition in reproductive success of RKN. Our results suggest that cystatins can play an important role for improving nematode resistance in eggplant and their deployment in gene pyramiding strategies with other proteinase inhibitors could ultimately enhance crop yield.

RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita

The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita -specific pioneer effector genes, such as msp-18 , msp-20 , msp-24 , msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa . Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita . This remarkable finding may provide some useful links for future research on nematode effector interaction.

An insight into the expression profile of defence-related genes in compatible and incompatible Oryza sativa-Meloidogyne graminicola interaction

The present study was conducted to investigate the comparative parasitic ability of root-knot nematode (RKN: Meloidogyne graminicola) in putatively resistant (Suraksha) and susceptible (Pusa 1121) cultivars of rice and to analyse the relative expression level of plant defence genes upon RKN infection in these cultivars. qRT-PCR analyses of RKNinfected roots and their corresponding shoots indicated that phytohormone-mediated host defences were activated during early infection in both susceptible and resistant plants, whereas it was apparently suppressed during later stage of infection in susceptible plants. Expression of defence genes in the shoot tissues of resistant plants suggested the successful induction of systemic acquired resistance. In parallel, lignin-and callose-mediated plant basal defence was activated in Suraksha. Information generated on the RKN-induced expression of defence genes in different rice varieties can be exploited to improve resistance in cultivated species of rice to RKN infection.

Molecular characterization of FMRFamide-like peptides in Meloidogyne graminicola and analysis of their knockdown effect on nematode infectivity

The rice root-knot nematode, Meloidogyne graminicola, seriously impairs the growth and yield of rice which is an important staple food worldwide. The disruption of neuropeptide signalling leading to attenuation in nematode behaviour and thereby perturbed infection, offers an attractive alternative to control nematodes. In this direction, the present study was aimed at mining of putative FMRFamide-like peptides (FLPs) from the transcriptomic dataset of M. graminicola followed by characterization of those FLPs via sequencing of PCR products, qRT-PCR and Southern hybridization analysis. We have characterized nine flp genes (flp-1, flp-3, flp-6, flp-7, flp-11, flp-12, flp-14, flp-16 and flp-18) and a partial neuropeptide receptor gene (flp-18 GPCR) from M. graminicola in the present study. In addition, in situ localization revealed the expression of flp-1 and flp-7 in neurons posterior to the circumpharyngeal nerve ring of M. graminicola. In vitro silencing of nine flp genes and flp-18 GPCR in M. graminicola J2 and their subsequent infection in rice and wheat roots demonstrated the reduced penetration ability of FLP silenced worms which underscores the potential of the FLPergic system as a broad-spectrum target to manage the root-knot nematode problem in rice-wheat cropping system.

Estimation of lipid reserves in different life stages of Meloidogyne incognita using image analysis of Nile Red-stained nematodes

Biochemical analyses of nematodes have revealed that neutral lipids (especially triglycerides) are the main source of energy reserves, which is depleted as the nematodes age. Several methodologies have been developed to visualise triglyceride-rich fat stores in plant-parasitic nematodes using non-fluorescent, lipophilic dyes, such as Oil Red O. Here, we propose a robust and reproducible fluorescence-based Nile Red staining method (followed by image analysis) for rapid detection of neutral lipid droplets in Meloidogyne incognita . This unique lipophilic dye selectively fluoresces in red and green spectra in a lipid-rich environment. The neutral lipid content of M. incognita juveniles gradually diminished during different periods of food deprivation, and this was significantly correlated with reduction in parasitic success of M. incognita in eggplant. Additionally, variation in fat reserves in different developmental stages of M. incognita infecting adzuki bean was also demonstrated. This investigation may aid future metabolic research, including functional analysis of lipid regulatory genes in plant-parasitic nematodes.