Surekha Kundu

In-vitro characterization of the behaviour of Macrophomina phaseolina (Tassi) Goid at the rhizosphere and during early infection of roots of resistant and susceptible varieties of sesame

There are major gaps in our knowledge of the stages of infection in soil borne pathogens. Soil borne diseases, such as charcoal root rot caused by Macrophomina phaseolina on sesame, have been studied, yet due to the difficulty in observing fungal behaviour in the soil, there has been no detailed study of the infection events. Moreover no study has attempted to compare the infection events in roots of resistant versus susceptible hosts. We present the first ultra-structural report to characterize the behaviour of the fungus in the proximity of the root, the appearance of fungal hyphae on the surface of roots, microsclerotia formation on hyphal strands, early penetration events and subsequent infection processes of M. phaseolina in sesame. We observed distinct differences in fungal behaviour in the rhizosphere and during infection of susceptible and resistant varieties. This study also describes a framework for comparative experiments. The possible reasons for the difference in behaviour of M. phaseolina in the vicinity of and during infection of roots of resistant vs. susceptible varieties of sesame and its implications for disease resistance are discussed.

Overexpression of a New Osmotin-Like Protein Gene (SindOLP) Confers Tolerance against Biotic and Abiotic Stresses in Sesame

Osmotin-like proteins (OLPs), of PR-5 family, mediate defense against abiotic, and biotic stresses in plants. Overexpression in sesame of an OLP gene (SindOLP), enhanced tolerance against drought, salinity, oxidative stress, and the charcoal rot pathogen. SindOLP was expressed in all parts and localized to the cytosol. The transgenic plants recovered after prolonged drought and salinity stress, showing less electrolyte leakage, more water content, longer roots, and smaller stomatal aperture compared to control plants. There was an increase in osmolytes, ROS-scavenging enzymes, chlorophyll content, proline, secondary metabolites, and reduced lipid peroxidation in the transgenic sesame under multiple stresses. The OLP gene imparted increased tolerance through the increased expression of three genes coding for ROS scavenging enzymes and five defense-related marker genes functioning in the JA/ET and SA pathways, namely Si-Apetala2, Si-Ethylene-responsive factor, Si-Defensin, Si-Chitinase, and Si-Thaumatin-like protein were monitored. The transgenic lines showed greater survival under different stresses compared to control through the integrated activation of multiple components of the defense signaling cascade. This is the first report of transgenic sesame and first of any study done on defense-related genes in sesame. This is also the first attempt at understanding the molecular mechanism underlying multi-stress tolerance imparted by an OLP.

Mechanism of regulation of tomato TRN1 gene expression in late infection with tomato leaf curl New Delhi virus (ToLCNDV)

Tomato leaf curl disease caused by geminiviruses is manifested by curling and puckering of leaves and thickening of veins, resembling developmental defects. This is probably due to the long-term altered regulation of expression of development related gene(s). Our results show that in the infected leaves the transcript level of TORNADO1 (SlTRN1), a gene important for cell expansion and vein formation, increased significantly. SlTRN1 is transcribed from two start sites. The preferential usage of one start site governs its expression in viral-stressed plants. To investigate the role of specific promoter elements in mediating differential expression of SlTRN1, we performed SlTRN1 promoter analysis. The promoter-regulatory sequences harbor multiple W-boxes. The SlWRKY16 transcription factor actively interacts with one of the W-boxes. WRKY proteins are commonly induced by salicylic acid (SA), and consequently SA treatment increased transcript level of SlWRKY16 and SlTRN1. Further mutational analyses confirmed the role of W-boxes in mediating SlTRN1 induction during ToLCNDV infection or SA treatment. We postulate that the activation of SA pathway during stress-response in tomato induces WRKY16, which in turn modulates transcription of SlTRN1 gene. This study unravels the mechanism of regulation of a developmental gene during stress-response, which may affect the severity of symptoms.

Biotrophy-necrotrophy switch in pathogen evoke differential response in resistant and susceptible sesame involving multiple signaling pathways at different phases

Infection stages of charcoal rot fungus Macrophomina phaseolina in sesame revealed for the first time a transition from biotrophy via BNS (biotrophy-to-necrotrophy switch) to necrotrophy as confirmed by transcriptional studies. Microscopy using normal and GFP-expressing pathogen showed typical constricted thick intercellular bitrophic hyphae which gave rise to thin intracellular necrotrophic hyphae during BNS and this stage was delayed in a resistant host. Results also show that as the pathogen switched its strategy of infection, the host tailored its defense strategy to meet the changing situation. Less ROS accumulation, upregulation of ROS signaling genes and higher antioxidant enzyme activities post BNS resulted in resistance. There was greater accumulation of secondary metabolites and upregulation of secondary metabolite-related genes after BNS. A total of twenty genes functioning in different aspects of plant defense that were monitored over a time course during the changing infection phases showed a coordinated response. Experiments using phytohormone priming and phytohormone inhibitors showed that resistance resulted from activation of JA-ET signaling pathway. Most importantly this defense response was more prompt in the resistant than the susceptible host indicating that a resistant host makes different choices from a susceptible host during infection which ultimately influences the severity of the disease.