Global transcriptome analysis reveals fungal disease responsive core gene regulatory landscape in tea.

Fungal infections are the inevitable limiting factor for productivity of tea. Transcriptome reprogramming recruits multiple regulatory pathways during pathogen infection. A comprehensive meta-analysis was performed utilizing previously reported, well-replicated transcriptomic datasets from seven fungal diseases of tea. The study identified a cumulative set of 18,517 differentially expressed genes (DEGs) in tea, implicated in several functional clusters, including the MAPK signaling pathway, transcriptional regulation, and the biosynthesis of phenylpropanoids. Gene set enrichment analyses under each pathogen stress elucidated that DEGs were involved in ethylene metabolism, secondary metabolism, receptor kinase activity, and various reactive oxygen species detoxification enzyme activities. Expressional fold change of combined datasets highlighting 2258 meta-DEGs shared a common transcriptomic response upon fungal stress in tea. Pervasive duplication events caused biotic stress-responsive core DEGs to appear in multiple copies throughout the tea genome. The co-expression network of meta-DEGs in multiple modules demonstrated the coordination of appropriate pathways, most of which involved cell wall organization. The functional coordination was controlled by a number of hub genes and miRNAs, leading to pathogenic resistance or susceptibility. This first-of-its-kind meta-analysis of host-pathogen interaction generated consensus candidate loci as molecular signatures, which can be associated with future resistance breeding programs in tea.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Comparative transcriptomic profiling of susceptible and resistant cultivars of pigeonpea demonstrates early molecular responses during Fusarium udum infection.

Vascular wilt caused by Fusarium udum Butler is the most important disease of pigeonpea throughout the world. F. udum isolate MTCC 2204 (M1) inoculated pigeonpea plants of susceptible (ICP 2376) and resistant (ICP 8863) cultivars were taken at invasion stage of pathogenesis process for transcriptomic profiling to understand defense signaling reactions that interplay at early stage of this plant-pathogen encounter. Differential transcriptomic profiles were generated through cDNA-AFLP from M1 inoculated resistant and susceptible pigeonpea root tissues. Twenty five percent of transcript derived fragments (TDFs) were found to be pathogen induced. Among them 73 TDFs were re-amplified and sequenced. Homology search of the TDFs in available databases and thorough study of scientific literature identified several pathways, which could play crucial role in defense responses of the F. udum inoculated resistant plants. Some of the defense responsive pathways identified to be active during this interaction are, jasmonic acid and salicylic acid mediated defense responses, cell wall remodeling, vascular development and pattering, abscisic acid mediated responses, effector triggered immunity, and reactive oxygen species mediated signaling. This study identified important wilt responsive regulatory pathways in pigeonpea which will be helpful for further exploration of these resistant components for pigeonpea improvement.

Elicitation of biomolecules as host defense arsenals during insect attacks on tea plants (Camellia sinensis (L.) Kuntze).

The most consumed and economically important beverage plant, tea (Camellia sinensis), and its pests have coevolved so as to maintain the plant-insect interaction. In this review, findings of different research groups on pest responsive tolerance mechanisms that exist in tea manifested through the production of secondary metabolites and their inducers are presented. The phytochemicals of C. sinensis have been categorized into volatiles, nonvolatiles, enzymes, and phytohormones for convenience. Two types of pests, namely the piercing-sucking pests and chewing pests, are associated with tea. Both the insect groups can trigger the production of those metabolites and inducers through several primary and secondary biosynthetic pathways. These induced biomolecules can act as insect repellents and most of them are associated with lowering the nutrient quality of plant tissue and increasing the indigestibility in the pest's gut. Moreover, some of them also act as predator attractants of particular pests. The herbivore-induced plant volatiles secreted from tea plants during pest infestation were (E)-nerolidol, α-farnesene, (Z)-3-hexenol, (E)-4,8-dimethyl-1,3,7-nonatriene, indole, benzyl nitrile (BN), linalool, and ocimenes. The nonvolatiles like theaflavin and L-theanine were increased in response to the herbivore attack. Simultaneously, S-adenosyl-L-methionine synthase, caffeine synthase activities were affected, whereas flavonoid synthesis and wax formation were elevated. Defense responsive enzymes like peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, ascorbate peroxidase, and catalase are involved in pest prevention mechanisms. Phytohormones like jasmonic acid, salicylic acid, abscisic acid, and ethylene act as the modulator of the defense system. The objective of this review is to discuss the defensive roles of these metabolites and their inducers against pest infestation in tea with an aim to develop environmentally sustainable pesticides in the future.Key points• Herbivore-induced volatile signals and their effects on neighboring tea plant protection• Stereochemical conversion of volatiles, effects of nonvolatiles, expression of defense-responsive enzymes, and phytohormones due to pest attack• Improved understanding of metabolites for bio-sustainable pesticide development.

Embryonic Explant and Plumular Meristem Transformation Methods for Development of Transgenic Pigeon Pea.

A reliable pigeon pea transformation system can assist the rapid improvement of this important grain legume through transgenic development. Here we describe two methods of Agrobacterium tumefaciens-mediated pigeon pea transformation. In the tissue culture based embryonic explant transformation method, microshoot grafting was included to obtain rapid root induction, while the other method was culture independent and designated as plumular meristem transformation. Both methods drastically enhanced the transformation frequency and have the potential to provide reasonable solutions for maximum transgenic recovery in biotechnological breeding programs.

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.