The biotechnological application and limitation of IRES to deliver multiple defence genes to plant pathogens

摘要

Transgenic resistance often has enhanced efficacy when more than one transgene is expressed. Here we explore the co-delivery of multiple discrete effectors via a single transgene using an internal ribosome entry site (IRES) sequence. As an example, we report the co-delivery of two distinct proteinase inhibitors in Nicotiana tabacum var. Xanthi from a bicistronic plant mRNA to examine resistance against plant parasitic nematodes. A cysteine proteinase inhibitor, Oc-IDeltaD86, is translated in a normal cap-dependent manner while translation of the serine proteinase inhibitor, CpTI, from the bicistronic mRNA is IRES-mediated. ELISAs using antibodies confirm the expression of the two inhibitors in aerial and root material and suggest that IRES-mediated expression in the roots is lower than normal cap-dependent expression. Analysis of Globodera tabacum recovered from transgenic Nicotiana expressing two discrete proteinase inhibitors revealed appreciable levels of resistance of up to 51 +/- 3%. Histochemical analysis of animals recovered from transgenic Nicotiana lines expressing Oc-IDeltaD86, via cap-dependent translation revealed a marked reduction in cysteine proteolyti,c activity in comparison to those from control untransformed plants. A less dramatic reduction was observed in similar analysis of serine proteolytic activity of animals recovered from transgenic Nicotiana lines expressing CpTI via IRES-mediated translation. The utility of using an IRES element to deliver a number of discrete anti-pathogen proteins is discussed.