Ramcharan Bhattacharya

Three hydroxyproline-rich glycopeptides derived from a single petunia polyprotein precursor activate defensin I, a pathogen defense response gene

Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16–20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18–21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.

Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae

Background Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated. Methodology/Principal Findings Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population. Conclusions/Significance The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids.

Isolation and Characterization of Hydroxyproline-Rich Glycopeptide Signals in Black Nightshade Leaves

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.

Aphid resistance in Brassica crops: challenges, biotechnological progress and emerging possibilities.

Aphids, (Hemiptera: Aphidoidea) a nefarious insect pest of Brassicaceae members including major vegetable and oilseed crops have coevolved with their host plant and emerged as most economically important insect pest of crop Brassicas. Their atypical feeding mechanism and unusual reproductive biology made them intractable to control below economic threshold level of damage to the crops. To a large extent aphid infestation is controlled by spraying agrochemicals of systemic mode of action and rarely by biological control. Use of systemic insecticides is highly cost intensive as well poses bigger threat of their incorporation in dietary chain. Breeding for genetic resistance against aphids has not been possible owing to the non-availability of resistance source within the crossable germplasms and lack of knowledge of the genetics of the trait. Genetic engineering with insect resistant transgenes seems to be the only potential avenue to address this difficult-to-accomplish breeding objective. Some success had been achieved in terms of developing aphid resistant cultivars through genetic engineering however, commercial utilization of such crops are still awaited. Thus protection of crops against aphids necessarily requires more research to identify either more effective insecticidal transgenes or biological phenomenon that can usher to new mechanism of resistance. The present review is an attempt to highlight the current status and possible avenues to develop aphid resistance in Brassicaceae crops.

Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

Hydroxyproline-rich glycopeptide signals in potato elicit signalling associated with defense against insects and pathogens.

HypSys peptides are 18-20 amino acids glycopeptide defense signal first discovered in tobacco and tomato that activate expression of defensive genes against insect-herbivores. Discovery of their orthologs in other Solanaceaous and nonsolanaceous plants demonstrated their possible ubiquitous nature and species specific functional diversity. In our continued search to establish the paradigm of defense signalling by HypSys peptides, we isolated a cDNA from potato leaves encoding putative analogs of tomato HypSys peptides flanked by conserved proteolytic cleavage sites. The gene encoding the cDNA was a member of a gene family in the tetraploid genome of potato and its expression was transcriptionally activated by wounding and methyl jasmonate. The deduced precursor protein contained a leader peptidase splice site and three putative HypSys peptides with conserved N- and C-termini along with central proline-rich motifs. In defense signalling, the three HypSys peptides elicit H₂O₂ generation in vivo and activate several antioxidant defensive enzymes in young potato leaves. Similar to potato systemin, the HypSys peptides activate the expression of octadecanoid pathway genes and protease inhibitors for insect defense. In addition, the HypSys peptides also activate the essential genes of the innate pathogen defense response in young potato leaves, acting as common elicitors of signalling associated with anti-herbivore and anti-pathogen defense in potato.

Peptide signals for plant defense display a more universal role

Hydroxyproline-rich systemins (HypSys) are small defense signaling glycopeptides found within the Solanaceae family that until recently were thought to only induce defense genes to herbivore attack. The glycopeptides are processed from larger proproteins with up to 3 different glycopeptides being processed out of a single precursor protein. A conserved central hydroxyproline motif within each HypSys is the site of pentose sugar attachment. Recently, it was found that in Petunia hybrida, these defense signaling glycopeptides did not induce protease inhibitor but instead, increased levels of defensin, a gene that is involved in pathogen attack. More recently, a HypSys peptide was isolated from Ipomoea batatas (sweet potato) of the Convolvulaceae family and found to induce sporamin. The proprotein precursor contained six putative peptide signals and had a propeptidase processing region with homology to solanaceous proHypSys. Thus, the HypSys defense peptides are no longer confined to defense against herbivory or exclusivity to the Solanaceae family, redefining both function and dispersion. Addendum to: Chen Y-C, Siems WF, Pearce G, Ryan CA. Six peptide wound signals derived from a single precursor protein in Ipomoea batatas leaves activate the expression of the defense gene sporamin. J Biol Chem 2008; 283:11469-76.

Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2.

BACKGROUND AND AIMS Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae. METHODS Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay. KEY RESULTS Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure. CONCLUSIONS Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of arabidopsis.

Host-mediated RNA interference targeting a cuticular protein gene impaired fecundity in the green peach aphid Myzus persicae : RNAi-mediated aphid resistance

BACKGROUND The green peach aphid (Myzus persicae) is a devastating sap-sucking insect pest that damages many host plants worldwide and causes billions of dollars of crop losses. Induction of RNA interference (RNAi) through oral feeding of small interfering RNA (siRNA) has been demonstrated in aphids. Therefore, host-mediated delivery of double-stranded RNA (dsRNA) specific to vital structural genes of aphids has been envisaged as a tool for the development of resistance against this aphid species. RESULTS Cuticular protein (CP) senses seasonal photoperiodism and drives a shift from clonal to sexual generation in aphids. Thus, attenuation of CP gene expression is likely to result in a different reproductive orientation in aphids and thereby affect their fecundity. A gene encoding CP in M. persicae has been targeted for RNAi-mediated knockdown. Transgenic Arabidopsis expressing dsRNA homologous to the MyCP gene was developed. The dsRNA-transgenics produced gene-specific siRNAs fed by aphids infesting the transgenics. A reverse transcription-quantitative polymerase chain reaction (RT-qPCR) study revealed an attenuated level of transcripts of the CP gene in aphid nymphs reared on the transgenic plants. Decreased expression of the CP gene resulted in a noticeable decline in aphid fecundity on the transgenic Arabidopsis plants. CONCLUSION Increasing genetic resistance is the only sustainable way of minimizing the use of toxic agrochemicals to protect plants. Host-mediated RNAi of important insect genes has been proposed as a potential avenue for developing crop resistance against insect pests. This study demonstrated the potential of MyCP dsRNA in developing RNAi-based resistance to M. persicae. RNAi-mediated resistance is expected to be more durable compared with other transgenic strategies.

RNAi for Resistance Against Biotic Stresses in Crop Plants

RNA interference (RNAi)-based gene silencing has become one of the most successful strategies in not only identifying gene function but also in improving agronomical traits of crops by silencing genes of different pathogens/pests and also plant genes for improvement of desired trait. The conserved nature of RNAi pathway across different organisms increases its applicability in various basic and applied fields. Here we attempt to summarize the knowledge generated on the fundamental mechanisms of RNAi over the years, with emphasis on insects and plant-parasitic nematodes (PPNs). This chapter also reviews the rich history of RNAi research, gene regulation by small RNAs across different organisms, and application potential of RNAi for generating transgenic plants resistant to major pests. But, there are some limitations too which restrict wider applications of this technology to its full potential. Further refinement of this technology in terms of resolving these shortcomings constitutes one of the thrust areas in present RNAi research. Nevertheless, its application especially in breeding agricultural crops resistant against biotic stresses will certainly offer the possible solutions for some of the breeding objectives which are otherwise unattainable.