Functional characterization of two banana NPR1 genes for pathogen defense response in Arabidopsis

Abstract

The Non-expressor of pathogenesis-related1 gene (NPR1) mediates the induction of pathogenesis-related (PR) gene products, vital for resistance in plants. In this study, the role of two previously isolated Cavendish banana NPR1-like genes (MNPR1A and MNPR1B) has been characterized in protection against Xanthomonas campestris, Hylaperonospora arabidopsidis, Botrytis cinerea and Pseudomonas syringae pathogens. The specific aim was to investigate if sequence differences in both genes are responsible for differential activity against pathogens because in a previous expression study, MNPR1A and not MNPR1B had been more responsive to the banana necrotrophic pathogen Fusarium oxysporum. By challenging Fusarium-tolerant GCTCV-218 and susceptible Grand Naine Cavendish banana plants (which had been used in a previous characterization study) with the hemi-biotrophic Xanthomonas pathogen (a very important economical pathogen of banana), the two MNPR1, PR-1 and PR-3 genes were found to be sequentially expressed. Expression of these genes was more pronounced in the tolerant GCTCV-218 banana cultivar than in the sensitive Grand Naine cultivar. Comparative sequence analysis further showed that these two banana NPR1-like coding sequences had dissimilarities even within conserved functional domains; they grouped closely with other defense-related NPR1-like sequences and harboured defense cis-regulatory elements. Transformation of the coding sequences of both genes under the control of the 35S CaMV promoter/terminator sequences into npr1-2 Arabidopsis mutant complimented the phenotype of this mutant following infection with distinct classes of pathogens (biotrophic Hyaloperonospora, necrotrophic Botrytis and hemi-biotrophic Pseudomonas pathogens). These Infected-MNPR1-expressing plants had higher PR-1 transcript amounts with more reduced pathogen growth compared to non-transgenic npr1-2 Arabidopsis mutant plants. However, the difference in the two banana coding sequences did not translate into a differential pattern of response against the three different classes of pathogens used in this study. Further detailed studies are suggested to investigate the role of the MNPR1 promoter-coding sequences in the differential response to pathogens using a bananapathogen system. This study also addressed the question of whether cystosolic glutathione (GSH) is necessary for NPR1 transcription during systemic acquired resistance. Using Arabidopsis mutants (clt1clt2clt3) defective in cytosolic GSH biosynthesis and following infection with either Pseudomonas or Botrytis, NPR1 and PR-1 transcription was much reduced rendering the mutants more sensitive to pathogens compared to infected-wild-type i>Arabidopsis plants. Results from this study therefore implicate cytosolic glutathione as an essential antioxidant for the establishment of an effective defense response cascade.