Comparative Analysis Delineates the Transcriptional Resistance Mechanisms for Pod Borer Resistance in the Pigeonpea Wild Relative Cajanus scarabaeoides (L.) Thouars.

Insect pests pose a serious threat to global food production. Pod borer (Helicoverpa armigera (Hübner)) is one of the most destructive pests of leguminous crops. The use of host resistance has been an effective, environmentally friendly and sustainable approach for controlling several agricultural pests. The exploitation of natural variations in crop wild relatives could yield pest-resistant crop varieties. In this study, we used a high-throughput transcriptome profiling approach to investigate the defense mechanisms of susceptible cultivated and tolerant wild pigeonpea genotypes against H. armigera infestation. The wild genotype displayed elevated pest-induced gene expression, including the enhanced induction of phytohormone and calcium/calmodulin signaling, transcription factors, plant volatiles and secondary metabolite genes compared to the cultivated control. The biosynthetic and regulatory processes associated with flavonoids, terpenes and glucosinolate secondary metabolites showed higher accumulations in the wild genotype, suggesting the existence of distinct tolerance mechanisms. This study provides insights into the molecular mechanisms underlying insect resistance in the wild pigeonpea genotype. This information highlights the indispensable role of crop wild relatives as a source of crucial genetic resources that could be important in devising strategies for crop improvement with enhanced pest resistance.

Comparative transcriptome analyses provide novel insights into the differential response of Pigeonpea (Cajanus cajan L.) and its wild relative (Cajanus platycarpus (Benth.) Maesen) to herbivory by Helicoverpa armigera (Hübner).

KEY MESSAGE: Deeper insights into the resistance response of Cajanus platycarpus were obtained based on comparative transcriptomics under Helicoverpa armigera infestation. Devastation by pod borer, Helicoverpa armigera is one of the major factors for stagnated productivity in Pigeonpea. Despite possessing a multitude of desirable traits including pod borer resistance, wild relatives of Cajanus spp. have remained under-utilized due to linkage drag and cross-incompatibility. Discovery and deployment of genes from them can provide means to tackle key pests like H. armigera. Transcriptomic differences between Cajanus platycarpus and Cajanus cajan during different time points (0, 18, 38, 96 h) of pod borer infestation were elucidated in this study. For the first ever time, we demonstrated captivating variations in their response; C. platycarpus apparently being reasonably agile with effectual transcriptomic reprogramming to deter the insect. Deeper insights into the differential response were obtained by identification of significant GO-terms related to herbivory followed by combined KEGG and ontology analyses. C. platycarpus portrayed a multilevel response with cardinal involvement of SAR, redox homeostasis and reconfiguration of primary metabolites leading to a comprehensive defense response. The credibility of RNA-seq analyses was ascertained by transient expression of selected putative insect resistance genes from C. platycarpus viz., chitinase (CHI4), Alpha-amylase/subtilisin inhibitor (IAAS) and Flavonoid 3_5 hydroxylase (C75A1) in Nicotiana benthamiana followed by efficacy analysis against H. armigera. qPCR validated results of the study provided innovative insights and useful leads for development of durable pod borer resistance.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Analysis of variation in virulence of Beauveria bassiana against insect pests of pigeonpea using qPCR.

Beauveria bassiana is a broad spectrum microbial bioagent used for the control of agriculturally important insect pests. However, in our experiments, two virulent isolates of B. bassiana (B2 and B10) showed specific preference toward Maruca vitrata and Helicoverpa armigera of pigeonpea. To better understand this feature, we developed a qPCR assay to quantify the chitinase (virulence factor) of B. bassiana during the infection process. Isolates of B. bassiana were grown on insect cuticle amended medium and minimal medium (without insect cuticle) to assess the induction of chitinase. Our results revealed a positive correlation between expression of chitinase by B. bassiana and the substrates (with or without cuticles of M. vitrata and H. armigera) used. This study showcases the methodology to quantify the chitinase and analysis of variation in virulence of B. bassiana (B2 and B10) against M. vitrata and H. armigera.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

KEY MESSAGE: Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Induced resistance by oxidative shifts in pigeonpea (Cajanus cajan L.) following Helicoverpa armigera (Hübner) herbivory.

BACKGROUND: Oxidative responses in leaves, developing seeds and the pod wall of nine pigeonpea genotypes were investigated against Helicoverpa armigera feeding. Out of nine genotypes, four were moderately resistant, three were intermediate and two were moderately susceptible genotypes. RESULTS: A significant shift in the oxidative status of pigeonpea following herbivory was depicted by the upregulation of diamine oxidase (DAO), polyamine oxidase (PAO) and lipoxygenase 2 (LOX 2) activities. Polyphenol oxidase (PPO) activity was significantly higher in the infested pod wall and leaves of moderately resistant genotypes than in those of moderately susceptible genotypes. H. armigera infestation markedly enhanced phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) activities in wounded tissues. The decline in ascorbate peroxidase (APX) activity and ascorbate content was lower in moderately resistant genotypes than in moderately susceptible genotypes. A significant decrease in LOX 3 activity was also observed in the infested pod wall of moderately resistant and intermediate genotypes. A lower malondialdehyde (MDA) content and higher proline content of the infested pod wall and developing seeds was observed. Higher activities of PPO, PAL and proline content in leaves of uninfested moderately resistant genotypes could either be an unrelated observation or alternatively could help in identifying H. armigera-resistant genotypes. CONCLUSION: The increase in activities of PPO, DAO, PAO, PAL and TAL and higher proline and lower MDA content upon herbivory suggested their integrated contribution in providing resistance to pigeonpea against H. armigera.

Nitrate reductase and nitrite as additional components of defense system in pigeonpea (Cajanus cajan L.) against Helicoverpa armigera herbivory.

Amylase inhibitors serve as attractive candidates of defense mechanisms against insect attack. Therefore, the impediment of Helicoverpa armigera digestion can be the effective way of controlling this pest population. Nitrite was found to be a potent mixed non-competitive competitive inhibitor of partially purified α-amylase of H. armigera gut. This observation impelled us to determine the response of nitrite and nitrate reductase (NR) towards H. armigera infestation in nine pigeonpea genotypes (four moderately resistant, three intermediate and two moderately susceptible). The significant upregulation of NR in moderately resistant genotypes after pod borer infestation suggested NR as one of the factors that determine their resistance status against insect attack. The pod borer attack caused greater reduction of nitrate and significant accumulation of nitrite in moderately resistant genotypes. The activity of nitrite reductase (NiR) was also enhanced more in moderately resistant genotypes than moderately susceptible genotypes on account of H. armigera herbivory. Expression of resistance to H. armigera was further revealed when significant negative association between NR, NiR, nitrite and percent pod damage was observed. This is the first report that suggests nitrite to be a potent inhibitor of H. armigera α-amylase and also the involvement of nitrite and NR in providing resistance against H. armigera herbivory.

Protease inhibitor (PI) mediated defense in leaves and flowers of pigeonpea (protease inhibitor mediated defense in pigeonpea).

More than 200 insect pests are found growing on pigeonpea. Insects lay eggs, attack and feed on leaves, flowers and developing pods. Plants have developed elaborate defenses against these insect pests. The present work evaluates protease inhibitor (PI) based defense of pigeonpea in leaves and flowers. PIs in the extracts of these tender tissues were detected by using gel X-ray film contact print method. Up to three PIs (PI-3, PI-4 and PI-5) were detected in these tissues as against nine (PI-1-PI-9) in mature seeds. PI-3 is the major component of these tissues. Mechanical wounding, insect chewing, fungal pathogenesis and application of salicylic acid induced PIs in pigeonpea in these tissues. Induction was found to be local as well as systemic but local response was stronger than systemic response. During both local and systemic induction, PI-3 appeared first. In spite of the presence and induction of PIs in these tender tissues and seeds farmers continue to suffer yield loses. This is due to the weak expression of PIs. However the ability of the plant to respond to external stimuli by producing defense proteins does not seem to be compromised. This study therefore indicates that PIs are components of both constitutive and inducible defense and provide a ground for designing stronger inducible defense (PIs or other insect toxin based) in pigeonpea.

Field screening and evaluation of pigeonpea genotypes against podborer (Helicoverpa armigera).

Hence an experiment was conducted to screen fifteen germplasm lines for their resistance/tolerance to podborer under natural infestation in pesticide-free open field. To asses the degree of infestation 250 green pods were randomly picked up from all the plants of each plot. The pods damaged by gram podborer were detected by the presence large bored holes in pods. Such infested and healthy pods were counted separately. The pod damage (%) was calculated on the basis of number of pods examined and the number of infested pods. Marketable green pod yield of each genotype under natural pest infestation was also recorded. On the basis of mean infestation, ICP13201 showed the lowest (25%) pod damage and showed lowest susceptibility among the genotypes studied. It was followed by ICP13208 and ICP11964 showed lower pod damage. The rest of the genotype suffered higher pod damage to Helicoverpa armigera. Considering the yield potential ICP13201, ICP13214 and ICP13212 showed higher yield potential than other genotypes. Considering the lower susceptibility to Helicoverpa armigera and higher yield potential ICP13201 was found to be the best.