Abstract
Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.
Highlights
Designing of pathogen-resistant plants can help to alleviate global food crisis
The transient activities of each of the sixteen 5 and 3 -end deletion constructs coupled to GUS reporter (Figure 1A) were evaluated in tobacco protoplasts
The data obtained clearly suggests that the relative GUS activity of the CsVMV8 (−215 to +166) was almost equivalent (∼1.1 times) to that obtained for the CaMV35S promoter; while the CsVMV7 (−256 to +166) ranked second
Summary
Designing of pathogen-resistant plants can help to alleviate global food crisis. An annual crop loss of 10% occurs worldwide due to fungal diseases; fungicides are the most commonly used means to prevent the colonization, sporulation, and growth of phytopathogenic fungi (Hahn, 2014; Drenth and Guest, 2016). On the other hand, causes epidemic disease development in cabbage, Tasmanian pyrethrum and oilseed rape (West et al, 2001; Pethybridge et al, 2005; Dilmaghani et al, 2009). It has devastating effects on field peas when associated with Ascochyta (Liu et al, 2016). Both Alternaria and Phoma were found to develop resistance against QoI or strobilurins and benzimidazole group of fungicides, respectively (Van de Graaf et al, 2003; Karaoglanidis et al, 2011; Kim et al, 2017). There is an urgent need to explore new alternatives that can mount a broader resistance spectrum as well as stable pattern of inheritance in the resistant plants (Russell, 2013)
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