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

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. Grand Naine and cv. Rasthali 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, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 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.

Characterization of Fusarium wilt resistant somaclonal variants of banana cv. Rasthali by cDNA-RAPD.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is counted among the most destructive diseases of crop plants in India. In the absence of any credible control measure to manage this disease, development of resistant cultivars is the best option. Somaclonal variations arising out of long term in vitro culture of plant tissues is an important source of genetic variability and the selection of somaclones having desired characteristics is a promising strategy to develop plants with improved characters. In the present study, we isolated a group of somaclonal variants of banana cv. Rasthali which showed efficient resistance towards Foc race 1 infection in repeated bioassays. cDNA-RAPD methodology using 96 decamer primers was used to characterize these somaclonal variants. Among the four differentially amplified bands obtained, one mapping to the coding region of a lipoxygenase gene was confirmed to be down regulated in the somaclones as compared to controls by real-time quantitative RT-PCR. Our results correlated well with earlier studies with lipoxygenase mutants in maize wherein reduced expression of lipoxygenase led to enhanced resistance towards Fusarium infection.

Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

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

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.

Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses.

Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause-effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse-maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress-inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.

Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection.

The banana aphid-transmitted Banana bunchy top virus (BBTV) is the most destructive viral pathogen of bananas and plantains worldwide. Lack of natural sources of resistance to BBTV has necessitated the exploitation of proven transgenic technologies for obtaining BBTV-resistant banana cultivars. In this study, we have explored the concept of using intron-hairpin-RNA (ihpRNA) transcripts corresponding to viral master replication initiation protein (Rep) to generate BBTV-resistant transgenic banana plants. Two ihpRNA constructs namely ihpRNA-Rep and ihpRNA-ProRep generated using Rep full coding sequence or Rep partial coding sequence together with its 5' upstream regulatory region, respectively, and castor bean catalase intron were successfully transformed into banana embryogenic cells. ihpRNA-Rep- and ihpRNA-ProRep-derived transgenic banana plants, selected based on preliminary screening for efficient reporter gene expression, were completely resistant to BBTV infection as indicated by the absence of disease symptoms after 6 months of viruliferous aphid inoculation. The resistance to BBTV infection was also evident by the inability to detect cDNAs coding for viral coat protein, movement protein and Rep protein by RT-PCR from inoculated transgenic leaf extracts. Southern analysis of the two groups of transgenics showed that ihpRNA transgene was stably integrated into the banana genome. The detection of small interfering RNAs (siRNAs) derived from the ihpRNA transgene sequence in transformed BBTV-resistant plants positively established RNA interference as the mechanism underlying the observed resistance to BBTV. Efficient screening of optimal transformants in this vegetatively propagated non-segregating fruit crop ensured that all the transgenic plants assayed were resistant to BBTV infection.