Petunia Floral Defensins with Unique Prodomains as Novel Candidates for Development of Fusarium Wilt Resistance in Transgenic Banana Plants

Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C- terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium–mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana

Molecular analysis of putatively transgenic banana plants.

<p>(A) Genomic DNA<i>-</i>PCR analysis of untransformed control (UC) and the transgenic lines (W2, W3, W8 and W22). (B) Southern blot analysis of p1301-<i>MusaWRKY71</i> 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 <i>MusaWRKY71</i> overexpression in transgenic banana lines. All gene expression values have been normalized against <i>Musa EF1α</i> cDNA expression levels. Expression of <i>MusaWRKY71</i> in untransformed plants has been assumed to be 1 for estimating the level of overexpression of <i>MusaWRKY71</i> in different transgenic lines. Values are mean ± SE.</p

<i>MusaWRKY71</i> Overexpression in Banana Plants Leads to Altered Abiotic and Biotic Stress Responses

<div><p>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 <i>WRKY</i> gene from banana, <i>MusaWRKY71</i>, which was inducible in response to a wide array of abiotic or biotic stress stimuli. The present work details the effects of <i>MusaWRKY71</i> overexpression in transgenic banana plants. Stable integration and overexpression of <i>MusaWRKY71</i> in transgenic banana plants was proved by Southern blot analysis and quantitative real time PCR. Transgenic banana plants overexpressing <i>MusaWRKY71</i> 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 <i>WRKY</i>, <i>pathogenesis-related (PR) protein genes</i>, <i>non-expressor of pathogenesis-related genes 1</i> (<i>NPR1</i>) and <i>chitinase</i> families analyzed, 10 genes (six belonging to <i>WRKY</i> family, three belonging to <i>PR proteins</i> family and one belonging to <i>chitinase</i> family) showed significant differential regulation in <i>MusaWRKY71</i> overexpressing lines. These results indicate that <i>MusaWRKY71</i> is an important constituent in the transcriptional reprogramming involved in diverse stress responses in banana.</p></div

Detached leaf oxidative stress assay of p1301-<i>MusaWRKY71</i> transgenic plants.

<p>(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-<i>MusaWRKY71</i> transgenic leaves exposed to methyl viologen. (C) MDA levels in untransformed and p1301-<i>MusaWRKY71</i> transgenic leaves exposed to methyl viologen.</p

Detached leaf salt stress assay of p1301-<i>MusaWRKY71</i> transgenic plants.

<p>(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-<i>MusaWRKY71</i> transgenic leaves exposed to salt. (C) MDA levels in untransformed and p1301-<i>MusaWRKY71</i> transgenic leaves exposed to salt.</p

Differential regulation of putative MusaWRKY71 downstream genes in <i>MusaWRKY71</i> overexpressing plants.

<p>Six <i>WRKY</i> genes (GSMUA_Achr4G02800_001, GSMUA_Achr7G14140_001, GSMUA_Achr10G06050_001, GSMUA_Achr7G25400_001, GSMUA_Achr4G07230_001, GSMUA_Achr4G03660_001), 3 <i>PR protein</i> genes (GSMUA_Achr6G17070_001, GSMUA_Achr4G23100_001, GSMUA_Achr2G13240_001), and 1 <i>chitinase</i> gene (GSMUA_Achr3G26900_001) showed differential expression in the transgenic plants. All gene expression values have been normalized against <i>Musa EF1α</i> cDNA expression levels. The x-axis represents the expression level of <i>MusaWRKY71</i> in control conditions. Values are mean ± SE.</p

Small RNA Profiling of Two Important Cultivars of Banana and Overexpression of miRNA156 in Transgenic Banana Plants

<div><p>Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv. <i>Grand Naine</i> and cv. <i>Rasthali</i> leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, <i>MusamiRNA156</i> in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from <i>MusamiRNA156</i> gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.</p></div

In vitro bioassay for Foc resistance in transgenic banana plants.

<p>In vitro bioassay was performed wherein p<i>PhDef1</i>-1301 (A and B) and p<i>PhDef2</i>-1301 (C and D) transgenic plants and untransformed control banana plants (UC) were challenged using Foc spore suspension. Four weeks post inoculation, untransformed control plants died whereas transgenic plants survived and showed normal growth.</p

Ex vivo bioassay for Foc resistance in transgenic banana plants.

<p>p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transgenic plants and untransformed control banana plants hardened in a greenhouse for 2 months were challenged with Foc mass culture. External and internal symptoms of Foc infection on p<i>PhDef1</i>-1301 (A and B) and p<i>PhDef2</i>-1301 (C and D) transgenic and untransformed control banana plants (UC). Untransformed control plants showed yellowing of leaves and wilting whereas transgenic plants showed negligible infection. Corm sections of control plants showed intense discoloration whereas the transgenic plants showed significantly less discoloration of the corm tissue.</p

Statistics of the novel miRNAs in the two libraries used for target prediction.

<p>Statistics of the novel miRNAs in the two libraries used for target prediction.</p