Chitinase CHIT36 from Trichoderma harzianum Enhances Resistance of Transgenic Carrot to Fungal Pathogens

Abstract

Microbial endochitinase CHIT36 is one of the lytic enzymes secreted by Trichoderma harzianum that exhibits antifungal activity in vitro. To evaluate its activity when expressed in planta, a plasmid containing chit36 gene under the control of CaMV 35S promoter and the nptII selection gene were introduced into polyethylene glycol (PEG)-treated carrot (Daucus carota L.) protoplasts. The transgenic plants expressing CHIT36 were used in resistance assays to evaluate their susceptibility to fungal pathogens. Laboratorybased assays on detached leaves and petioles showed that the transgenic carrots had less severe disease symptoms. The resistance response depended on the transgenic clone, but all clones had significantly enhanced tolerance to Alternaria radicina and Botrytis cinerea, on average by 50%. Slower disease progress caused by Alternaria dauci was observed for two transgenic clones while the remaining clone was more susceptible than the control. The most resistant transgenic clones were also more tolerant to the pathogens than ÔBoleroÕ F 1 , which is a conventionally bred cultivar tolerant to A. dauci. This is the first report of the use of microbial chitinase to enhance carrot resistance. The results indicate that CHIT36 expressed in planta has the potential to reduce development of fungal diseases.

Defense-related gene expression and enzyme activities in transgenic cotton plants expressing an endochitinase gene from Trichoderma virens in response to interaction with Rhizoctonia solani

“…Expression of certain endochitinase encoding genes from Trichoderma spp. in plants has been shown to improve their defense response against various fungal pathogens ( Baranski et al 2008;Bolar et al 2000Bolar et al , 2001Dana et al 2006;Distefano et al 2008;Emani et al 2003;Lorito et al 1998). Previous work from our laboratory has demonstrated that cotton plants transformed with a gene encoding 42 kDa endochitinase from T. virens showed a high level of resistance to infection by Rhizoctonia solani and Alternaria alternata (Emani et al 2003).…”

Broad-spectrum disease resistance to necrotrophic and biotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1 gene

“…Genetic engineering for enhanced disease resistance in carrot has been previously shown to be partially successful through the expression of a single pathogenesis-related (PR) protein, such as chitinase, -1,3 glucanase, thaumatinlike protein or peroxidase (Punja and Raharjo 1996;Melchers and Stuiver 2000;Chen and Punja 2002;Punja 2005;Jayaraj and Punja 2007;Baranski et al 2008;Wally et al 2009), or using a combination of two genes (Melchers and Stuiver 2000;Jayaraj and Punja 2007;Wally et al 2009). These approaches have not, however, provided broad-spectrum resistance towards a wide range of pathogens.…”

Plant-beneficial effects of Trichoderma and of its genes

“…The homologous chit42 gene from T. virens was able to enhance resistance against R. solani when it was expressed in rice (Shah et al, 2009). Other Trichoderma chitinase genes have been used to generate transgenic plants resistant to fungal diseases: (i) tobacco cell cultures expressing the T. harzianum endochitinase chit40 gene released the enzyme into the medium and were able to inhibit the conidial germination of the post-harvest pathogen Penicillium digitatum (Brants & Earle, 2001); (ii) transgenic cotton plants expressing the T. virens endochitinase gene Tv-ech1 showed significant resistance to A. alternata and R. solani and a rapid and greater induction of ROS, followed by modulation of the expression of several defence-related genes and the induction of the terpenoid pathway (Emani et al, 2003;Kumar et al, 2009); (iii) the expression of the endochitinase chit36 gene of T. harzianum in carrot significantly enhanced tolerance to A. radicina and B. cinerea (Baranski et al, 2008).…”