Evaluation of expression of the Hrap and Pflp genes in transgenic banana plants for resistance against Xanthomonas campestris pv. musacearum

Abstract

Banana Xanthomonas wilt (BXW) remains a major problem to banana production in the East and Central African region. This PhD study evaluated the expression of stacked Hrap (hypersensitive response assisting protein) and Pflp (plant ferredoxin like protein) genes to possibly provide higher resistance to Xanthomonas campestris pv. musacearum (Xcm) compared to single gene expression. An expression plasmid vector having stacked Pflp and Hrap genes under control of a constitutive CaMV35s promoter was constructed using PCR cloning and restriction enzymes analysis. This plasmid vector was used to transform embryogenic cells using Agrobacterium tumefaciens transformation system and transgenic plants of banana cultivars Gonja manjaya and Sukali ndiizi were generated. Twenty one soil potted transgenic lines each of stacked genes as well as of the single Hrap or Pflp were artificially inoculated with Xcm and evaluated for resistance to BXW under screen house conditions. From inoculated plants, 28.6% of single Hrap lines, 19.1% of single Pflp lines and 33.3% of stacked lines had no disease symptoms and were completely resistant to BXW. Partial resistance was observed in 28.6% of single Hrap lines, 33.3% of single Pflp lines and 28.6% of stacked lines with only inoculated leaf showing symptoms. All non-transgenic control lines were susceptible and wilted completely. Also lesion length and chlorotic area were significantly smaller in all transgenic plants in comparison to non-transgenic control plants. However, no significant difference in lesion length and chlorotic area was observed transgenic lines with single Hrap and Pflp transgenes in comparison to lines stacked genes. Histochemical staining and calorimetric assays further confirmed higher hydrogen peroxide accumulation after Xcm infection in all transgenic plants which was related with increased transcription of NPR1 genes. NPR1 gene induction was also associated with transcription of PR1, PR3 and GST genes in transgenic banana lines. However, PR1 gene transcription was significantly lower in all transgenic lines than in the non-transgenic control plants. In contrast, transcription of PR3 gene, and also of a gene coding for GST, was higher in all transgenic lines than the non-transgenic control after Xcm infection. Hrap and Pflp transgene expression thereby shifted the pathogen response to a response normally expected for a necrotrophic pathogen. Overall, this study has provided evidence that stacked expression of Hrap and Pflp genes in transgenic banana lines did not have higher or additive resistance levels to Xcm compared to single genes. Future field studies should be carried out to demonstrate if developed transgenic lines expressing stacked transgenes have durable resistance to Xcm compared to lines expressing a single transgene.