Understanding sugarcane defence responses during the initial phase of Colletotrichum falcatum pathogenesis by suppression subtractive hybridization (SSH)

Abstract

Red rot caused by the fungus Colletotrichum falcatum Went is a destructive disease of sugarcane in India and other Asian countries. The pathogen infects the economically valuable stalk tissue which results in severe yield loss and impaired juice quality. Visible symptoms of the disease appear in the pathogen inoculated cane tissue after 72 h as reddish tissue discoloration. However, early molecular events that occur between the host and the pathogen before 72 h are poorly understood. To understand the molecular defense responses between sugarcane and C. falcatum interaction before phenotyphic symptom expression, we employed suppression subtractive hybridization (SSH) in sugarcane stalk tissue samples collected at 12 h and 36 h after pathogen inoculation in a red rot resistant cultivar. RNA samples of red rot resistant cv. Co 93009 collected at 12 h and 36 h after C. falcatum inoculation were used as a tester and the corresponding RNA samples of red rot susceptible cv.CoC 671 collected at 12 h and 36 h after pathogen inoculation and mock samples of cv. Co 93009 were used as drivers for the two individual subtractions. At the end of subtractive hybridization and sequencing, a total of 139 EST's were obtained from the two libraries which were functionally categorized as belonging to recognition and signal transduction, oxidative stress, redox maintenance, membrane trafficking and transport, defence and programmed cell death, energy and photosynthesis, metabolism, secondary metabolite biosynthesis, cell/nuclear structure and unknown categories. In 12 h response library (12hRL), the abundance of recognition and signal transduction ESTs were high whereas in 36 h response library (36hRL), ESTs homologous to nucleic acid metabolism were high. Further gene expression of a set of 12 candidate ESTs was validated by quantitative real-time PCR (qRT-PCR) in the same set of red rot resistant and susceptible cultivars. This study identified a network of early defence responses and associated signals for the first time in a red rot resistant sugarcane cultivar in response to C. falcatum infection.