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Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants.

ABSTRACT: In order to feed an ever-increasing world population, there is an urgent need to improve the production of staple food and fruit crops. The productivity of important food and fruit crops is constrained by numerous biotic and abiotic factors. The cultivation of banana, which is an important fruit crop, is severely threatened by Fusarium wilt disease caused by infestation by an ascomycetes fungus Fusarium oxysporum f. sp. cubense (Foc). Since there are no established edible cultivars of banana resistant to all the pathogenic races of Foc, genetic engineering is the only option for the generation of resistant cultivars. Since Foc is a hemibiotrophic fungus, investigations into the roles played by different cell-death-related genes in the progression of Foc infection on host banana plants are important. Towards this goal, three such genes namely MusaDAD1, MusaBAG1 and MusaBI1 were identified in banana. The study of their expression pattern in banana cells in response to Foc inoculation (using Foc cultures or fungal toxins like fusaric acid and beauvericin) indicated that they were indeed differentially regulated by fungal inoculation. Among the three genes studied, MusaBAG1 showed the highest up-regulation upon Foc inoculation. Further, in order to characterize these genes in the context of Foc infection in banana, we generated transgenic banana plants constitutively overexpressing the three genes that were later subjected to Foc bioassays in a contained greenhouse. Among the three groups of transgenics tested, transformed banana plants overexpressing MusaBAG1 demonstrated the best resistance towards Foc infection. Further, these plants also showed the highest relative overexpression of the transgene (MusaBAG1) among the three groups of transformed plants generated. Our study showed for the first time that native genes like MusaBAG1 can be used to develop transgenic banana plants with efficient resistance towards pathogens like Foc.

SUBMITTER: Ghag SB

PROVIDER: S-EPMC4122335 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants.

Ghag Siddhesh B SB Shekhawat Upendra K Singh UK Ganapathi Thumballi R TR

AoB PLANTS 20140704

In order to feed an ever-increasing world population, there is an urgent need to improve the production of staple food and fruit crops. The productivity of important food and fruit crops is constrained by numerous biotic and abiotic factors. The cultivation of banana, which is an important fruit crop, is severely threatened by Fusarium wilt disease caused by infestation by an ascomycetes fungus Fusarium oxysporum f. sp. cubense (Foc). Since there are no established edible cultivars of banana res ...[more]

PMID: 24996429

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Project description:BackgroundPlant microbiome highlights the importance of endosphere microbiome for growth and health of the host plant. Microbial community analysis represents an elegant way to identify keystone microbial species that have a more central position in the community. The aim of this study was to access the interactions between the keystone bacterial species and plants during banana Fusarium wilt process, by comparing the endophytic bacterial and fungal community in banana roots and shoot tips during growth and wilting processes. The keystone bacterial species were isolated and further engineered to improve banana wilt resistance.ResultsBanana endosphere microbiome structure varied during plant growth and wilting processes. Bacterial and fungal diversity in the shoot tips and roots increased with the development of the banana plantlets. The bacterial groups belonging to the Enterobacteriaceae family with different relative abundances were detected in all the samples. The Klebsiella spp. might be the keystone bacteria during the growth of banana plantlets. The relative abundance of Fusarium associated with the wilt disease did not increase during the wilting process. The endophytic Enterobacteriaceae strains Enterobacter sp. E5, Kosakonia sp. S1, and Klebsiella sp. Kb were isolated on Enterobacteriaceae selective medium and further engineered by expressing 1-aminocyclopropane-1-carboxylate (ACC) deaminase on the bacterial cell walls (designated as E5P, S1P, and KbP, respectively). Pot experiments suggested that plants inoculated with strains E5, E5P, S1, and S1P increased resistance to the Fusarium wilt disease compared with the controls without inoculation, whereas the Klebsiella inoculation (Kb and KbP) did not increase the wilt resistance. Compared with the inoculation with the wild strains E5 and S1, the inoculation with engineered strains E5P and S1P significantly increased wilt resistance and promoted plant growth, respectively. The results illustrated that the keystone species in the banana microbiome may not be dominant in numbers and the functional role of keystone species should be involved in the wilt resistance.ConclusionThe ACC deaminase activity of engineered bacteria was essential to the Fusarium wilt resistance and growth promotion of banana plants. Engineering keystone bacteria in plant microbiome with ACC deaminase on the cell walls should be a promising method to improve plant growth and disease resistance.

Project description:Iron is an indispensable element for plant growth and defense and hence it is essential to improve the plant's ability to accumulate iron. Besides, it is also an important aspect for human health. In view of this, we attempted to increase the iron content in banana cultivar Rasthali using MusaFer1 as a candidate gene. Initially, the expression of all five genes of the MusaFer family (MusaFer1-5) was quantified under iron-excess and -deficient conditions. The supplementation of 250 and 350 ?M iron enhanced expression of all MusaFer genes; however, MusaFer1 was increased maximally by 2- and 4- fold in leaves and roots respectively. Under iron deficient condition, all five MusaFer genes were downregulated, indicating their iron dependent regulation. In MusaFer1 overexpressing lines, iron content was increased by 2- and 3-fold in leaves and roots respectively, as compared with that of untransformed lines. The increased iron was mainly localized in the epidermal regions of petiole. The analysis of MusaFer1 promoter indicated that it might control the expression of iron metabolism related genes and also other genes of MusaFer family. MusaFer1 overexpression led to downregulated expression of MusaFer3, MusaFer4 and MusaFer5 in transgenic leaves which might be associated with the plant's compensatory mechanism in response to iron flux. Other iron metabolism genes like Ferric reductase (FRO), transporters (IRT, VIT and YSL) and chelators (NAS, DMAS and NAAT) were also differentially expressed in transgenic leaf and root, suggesting the multifaceted impact of MusaFer1 towards iron uptake and organ distribution. Additionally, MusaFer1 overexpression increased plant tolerance against methyl viologen and excess iron which was quantified in terms of photosynthetic efficiency and malondialdehyde content. Thus, the study not only broadens our understanding about iron metabolism but also highlights MusaFer1 as a suitable candidate gene for iron fortification in banana.

Project description:Fusarium wilt is currently spreading in banana growing regions around the world leading to substantial losses. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), which is further classified into distinct races according to the banana varieties that they infect. Cavendish banana is resistant to Foc race 1, to which the popular Gros Michel subgroup succumbed last century. Cavendish effectively saved the banana industry, and became the most cultivated commercial subgroup worldwide. However, Foc tropical race 4 (TR4) subsequently emerged in Southeast Asia, causing significant yield losses due to its high level of aggressiveness to cultivars of Cavendish, and other commonly grown cultivars. Preventing further spread is crucially important in the absence of effective control methods or resistant market-acceptable banana cultivars. Implementation of quarantine and containment measures depends on early detection of the pathogen through reliable diagnostics. In this study, we tested the hypothesis that secreted in xylem (SIX) genes, which currently comprise the only known family of effectors in F. oxysporum, contain polymorphisms to allow the design of molecular diagnostic assays that distinguish races and relevant VCGs of Foc. We present specific and reproducible diagnostic assays based on conventional PCR targeting SIX genes, using as templates DNA extracted from pure Foc cultures. Sets of primers specifically amplify regions of: SIX6 in Foc race 1, SIX1 gene in TR4, SIX8 in subtropical race 4, SIX9/SIX10 in Foc VCG 0121, and SIX13 in Foc VCG 0122. These assays include simplex and duplex PCRs, with additional restriction digestion steps applied to amplification products of genes SIX1 and SIX13. Assay validations were conducted to a high international standard including the use of 250 Fusarium spp. isolates representing 16 distinct Fusarium species, 59 isolates of F. oxysporum, and 21 different vegetative compatibility groups (VCGs). Tested parameters included inter and intraspecific analytical specificity, sensitivity, robustness, repeatability, and reproducibility. The resulting suite of assays is able to reliably and accurately detect R1, STR4, and TR4 as well as two VCGs (0121 and 0122) causing Fusarium wilt in bananas.

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Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants.

Publications

Native cell-death genes as candidates for developing wilt resistance in transgenic banana plants.

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