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. 2013 Oct 8;8(10):e75506.
doi: 10.1371/journal.pone.0075506. eCollection 2013.

MusaWRKY71 overexpression in banana plants leads to altered abiotic and biotic stress responses

Upendra K S Shekhawat  1 , Thumballi R Ganapathi
Affiliations

MusaWRKY71 overexpression in banana plants leads to altered abiotic and biotic stress responses

Upendra K S Shekhawat et al. PLoS One. .

Abstract

WRKY transcription factors are specifically involved in the transcriptional reprogramming following incidence of abiotic or biotic stress on plants. We have previously documented a novel WRKY gene from banana, MusaWRKY71, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of MusaWRKY71 overexpression in transgenic banana plants. Stable integration and overexpression of MusaWRKY71 in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing MusaWRKY71 displayed enhanced tolerance towards oxidative and salt stress as indicated by better photosynthesis efficiency (Fv/Fm) and lower membrane damage of the assayed leaves. Further, differential regulation of putative downstream genes of MusaWRKY71 was investigated using real-time RT-PCR expression analysis. Out of a total of 122 genes belonging to WRKY, pathogenesis-related (PR) protein genes, non-expressor of pathogenesis-related genes 1 (NPR1) and chitinase families analyzed, 10 genes (six belonging to WRKY family, three belonging to PR proteins family and one belonging to chitinase family) showed significant differential regulation in MusaWRKY71 overexpressing lines. These results indicate that MusaWRKY71 is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of transgenic banana cv. Rasthali plants overexpressing MusaWRKY71.
(A) T-DNA region of binary vector p1301-MusaWRKY71 designed to constitutively overexpress MusaWRKY71 in transgenic banana plants. (B) Transformed embryos on embryo induction medium. (C) Transgenic multiple shoots on multiple shoot induction medium. (D) Transgenic rooted plantlets on rooting medium. (E) Transgenic hardened plants in greenhouse (2-months old).
Figure 2
Figure 2. Molecular analysis of putatively transgenic banana plants.
(A) Genomic DNA-PCR analysis of untransformed control (UC) and the transgenic lines (W2, W3, W8 and W22). (B) Southern blot analysis of p1301-MusaWRKY71 transformed banana lines. (C) Real-time quantitative RT-PCR analysis of the selected transgenic lines (W2, W3, W8 and W22) for determination of the exact quantum of MusaWRKY71 overexpression in transgenic banana lines. All gene expression values have been normalized against Musa EF1α cDNA expression levels. Expression of MusaWRKY71 in untransformed plants has been assumed to be 1 for estimating the level of overexpression of MusaWRKY71 in different transgenic lines. Values are mean ± SE.
Figure 3
Figure 3. Detached leaf oxidative stress assay of p1301-MusaWRKY71 transgenic plants.
(A) Detached banana leaves derived from greenhouse maintained transgenic (W2, W3, W8 and W22) and control plants (UC) after exposure to simulated oxidative stress (10 µM methyl viologen in 1/10 MS basal medium for 7 days). (B) Photosynthetic efficiency (measured as Fv/Fm ratio) of untransformed and p1301-MusaWRKY71 transgenic leaves exposed to methyl viologen. (C) MDA levels in untransformed and p1301-MusaWRKY71 transgenic leaves exposed to methyl viologen.
Figure 4
Figure 4. Detached leaf salt stress assay of p1301-MusaWRKY71 transgenic plants.
(A) Detached banana leaves derived from greenhouse maintained transgenic (W2, W3, W8 and W22) and control plants (UC) after exposure to simulated salt stress (350 mM NaCl in 1/10 MS basal medium for 7 days). (B) Photosynthetic efficiency (measured as Fv/Fm ratio) of untransformed and p1301-MusaWRKY71 transgenic leaves exposed to salt. (C) MDA levels in untransformed and p1301-MusaWRKY71 transgenic leaves exposed to salt.
Figure 5
Figure 5. Differential regulation of putative MusaWRKY71 downstream genes in MusaWRKY71 overexpressing plants.
Six WRKY genes (GSMUA_Achr4G02800_001, GSMUA_Achr7G14140_001, GSMUA_Achr10G06050_001, GSMUA_Achr7G25400_001, GSMUA_Achr4G07230_001, GSMUA_Achr4G03660_001), 3 PR protein genes (GSMUA_Achr6G17070_001, GSMUA_Achr4G23100_001, GSMUA_Achr2G13240_001), and 1 chitinase gene (GSMUA_Achr3G26900_001) showed differential expression in the transgenic plants. All gene expression values have been normalized against Musa EF1α cDNA expression levels. The x-axis represents the expression level of MusaWRKY71 in control conditions. Values are mean ± SE.
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