Anita Grover

Taming abiotic stresses in plants through genetic engineering: current strategies and perspective

Abstract During the last decade, major advances have been made in plant genetic engineering. The methods for stable genetic transformation as well as for regulation of introduced trans-genes have been optimised to a great deal. The major limiting factor in the widespread application of genetic engineering is the availability of the target genes. This is particularly true for engineering tolerance against abiotic stresses (such as those caused by high levels of salts in soils, reduced/excess availability of water and sub- and supra-optimal temperature regimes). In spite of this, the past 5 years (1993–1998) have witnessed significant achievements in terms of generating transgenics for enhanced tolerance to these stresses. For future work on producing plants with still higher level of tolerance, there is a need to expand the information on the stress-induced genes so that appropriate genes can be pyramided. The current upsurge in genomic research has the potential to catalyse efforts in elucidating new stress-responsive genes. There are also possibilities of engineering the whole cascade of multiple genetic changes through manipulation of the regulatory genes.

Chitinase-mediated inhibitory activity of Brassica transgenic on growth of Alternaria brassicae.

Chitinase, capable of degrading the cell walls of invading phytopathogenic fungi, plays an important role in plant defense response, particularly when this enzyme is overexpressed through genetic engineering. In the present study, Brassica plant (Brassica juncea L.) was transformed with chitinase gene tagged with an overexpressing promoter 35 S CaMV. The putative transgenics were assayed for their inhibitory activity against Alternaria brassicae, the inducer of Alternaria leaf spot of Brassica both in vitro and under polyhouse conditions. In in vitro fungal growth inhibition assays, chitinase inhibited the fungal colony size by 12-56% over the non-trangenic control. The bioassay under artificial epiphytotic conditions revealed the delay in the onset of disease as well as reduced lesion number and size in 35S-chitinase Brassica as compared to the untransformed control plants.

Overexpression of NPR1 in Brassica juncea Confers Broad Spectrum Resistance to Fungal Pathogens

Brassica juncea (Indian mustard) is a commercially important oil seed crop, which is highly affected by many biotic stresses. Among them, Alternaria leaf blight and powdery mildew are the most devastating diseases leading to huge yield losses in B. juncea around the world. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the B. juncea disease resistance against these pathogens. NPR1 (non-expressor of pathogen-related gene 1) is a bonafide receptor of salicylic acid (SA) which modulates multiple immune responses in plants especially activation of induced and systemic acquired resistance (SAR). Here, we report the isolation and characterization of new NPR1 homolog (BjNPR1) from B. juncea. The phylogenetic tree constructed based on the deduced sequence of BjNPR1 with homologs from other species revealed that BjNPR1 grouped together with other known NPR1 proteins of Cruciferae family, and was nearest to B. napus. Furthermore, expression analysis showed that BjNPR1 was upregulated after SA treatment and fungal infection but not by jasmonic acid or abscisic acid. To understand the defensive role of this gene, we generated B. juncea transgenic lines overexpressing BjNPR1, and further confirmed by PCR and Southern blotting. The transgenic lines showed no phenotypic abnormalities, and constitutive expression of BjNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, BjNPR1 transgenic lines showed enhanced resistance to Alternaria brassicae and Erysiphe cruciferarum as there was delay in symptoms and reduced disease severity than non-transgenic plants. In addition, the rate of disease spreading to uninfected or distal parts was also delayed in transgenic plants thus suggesting the activation of SAR. Altogether, the present study suggests that BjNPR1 is involved in broad spectrum of disease resistance against fungal pathogens.

Expression analysis of chitinase upon challenge inoculation to Alternaria wounding and defense inducers in Brassica juncea

Highlights • Expression of chitinase gene was studied by RT-PCR in response to Alternaria brassicae.• Chitinase gene is induced by Alternaria, wounding and by JA and not by SA. It shows the tissue specificity of the gene.• Pathogen-inducible 2.5 kb chitinase class IV promoter was isolated from B. juncea by Genome Walking.• Induction pattern of chitinase gene is also reflected in promoter validation studied in transgenic Arabidopsis leaf.• This will help in using this promoter discretely in developing fungus resistant transgenic plants.

Removal of Vacuolar Targeting Signal from Class I Vacuolar Chitinase leads to its Extracellular Secretion in Transgenic Tobacco

Chitinase, an antifungal pathogenesis related (PR) protein is present in different isoforms. Class I basic chitinase which is generally more antifungal in nature compared to other chitinase classes, is present in vacuoles. It is speculated that extracellular secretion of this vacuolar enzyme by removing its vacuolar targeting signal at C- terminus might further increase its effectivity. Tobacco class I chitinase cDNA was earlier modified by PCR to add two stop codons before vacuolar targeting signal, so that the protein without this signal would be secreted extracellularly.Transgenic tobacco plants were raised with modified chitinase cDNA and native unmodified cDNA, both under the control of CaMV 35 S promoter, using Agrobacterium mediated transformation. Transgenic plants with unmodified class I chitinase cDNA expressed the enzyme in vacuoles and those having modified cDNA expressed the enzyme in extracellular spaces while retaining its biological activity.

Differential Induction of β-1,3 Glucanase in Resistant and Susceptible Varieties of Cicer arietinum L. Following Infection with Ascochyta rabiei

Infection of chickpea (Cicer arietinum L.) by Ascochyts rabiei resulted in marked induction of β-1, 3 glucanase activity. The induction of enzyme activity was two fold higher in resistant variety as compared to the susceptible variety. Slot blot analysis of total RNA using glucanase cDNA clone from tobacco as probe revealed that the induction of enzyme activity is at mRNA level.

Further studies on the metabolism of phospholipids inStreptomyces griseus: Turnover of phospholipid moieties

Phospholipid turnover inStreptomyces griseus was studied by pulse-chase techniques using 1-[14C]sodium acetate and [U-14C]glucose. Different phospholipids and their individual moieties were found to have different turnover rates. The moieties of inositol-containing phospholipids exhibited the fastest turnover rates among the major phospholipids, while only fatty acyl moieties of phosphatidylethanolamine showed rapid turnover. Cardiolipin did not show any significant turnover with both precursors.

Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance

Pathogenesis-related (PR) proteins and antimicrobial peptides (AMPs) are a group of diverse molecules that are induced by phytopathogens as well as defense related signaling molecules. They are the key components of plant innate immune system especially systemic acquired resistance (SAR), and are widely used as diagnostic molecular markers of defense signaling pathways. Although, PR proteins and peptides have been isolated much before but their biological function remains largely enigmatic despite the availability of new scientific tools. The earlier studies have demonstrated that PR genes provide enhanced resistance against both biotic and abiotic stresses, which make them one of the most promising candidates for developing multiple stress tolerant crop varieties. In this regard, plant genetic engineering technology is widely accepted as one of the most fascinating approach to develop the disease resistant transgenic crops using different antimicrobial genes like PR genes. Overexpression of PR genes (chitinase, glucanase, thaumatin, defensin and thionin) individually or in combination have greatly uplifted the level of defense response in plants against a wide range of pathogens. However, the detailed knowledge of signaling pathways that regulates the expression of these versatile proteins is critical for improving crop plants to multiple stresses, which is the future theme of plant stress biology. Hence, this review provides an overall overview on the PR proteins like their classification, role in multiple stresses (biotic and abiotic) as well as in various plant defense signaling cascades. We also highlight the success and snags of transgenic plants expressing PR proteins and peptides.