Transgenic East African Highland Banana Plants Are Protected against Radopholus similis through Host-Delivered RNAi.

The burrowing nematode Radopholus similis is considered a major problem of intensive banana cultivation. It can cause extensive root damage resulting in the toppling disease of banana, which means that plants fall to the ground. Soaking R. similis in double-stranded (ds) RNA of the nematode genes Rps13, chitin synthase (Chs-2), Unc-87, Pat-10 or beta-1,4-endoglucanase (Eng1a) suppressed reproduction on carrot discs, from 2.8-fold (Chs-2) to 7-fold (Rps13). The East African Highland Banana cultivar Nakitembe was then transformed with constructs for expression of dsRNA against the same genes, and for each construct, 30 independent transformants were tested with nematode infection. Four months after transfer from in vitro culture to the greenhouse, the banana plants were transferred to a screenhouse and inoculated with 2000 nematodes per plant, and thirteen weeks later, they were analyzed for several parameters including plant growth, root necrosis and final nematode population. Plants with dsRNA constructs against the nematode genes were on average showing lower nematode multiplication and root damage than the nontransformed controls or the banana plants expressing dsRNA against the nonendogenous gene. In conclusion, RNAi seems to efficiently protect banana against damage caused by R. similis, opening perspectives to control this pest.

First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA.

Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9-16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3-4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum, is the most devastating disease of banana in the Great Lakes region of Africa. The pathogen's rapid spread has threatened the livelihood of millions of Africans who rely on banana fruit for food security and income. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas and exotic types of banana. In the absence of natural host plant resistance among banana cultivars, the constitutive expression of the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper (Capsicum annuum) was evaluated for its ability to confer resistance to BXW. Transgenic lines expressing the Hrap gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of two banana cultivars: 'Sukali Ndiizi' and 'Mpologoma'. These lines were characterized by molecular analysis, and were challenged with Xanthomonas campestris pv. musacearum to analyse the efficacy of the Hrap gene against BXW. The majority of transgenic lines (six of eight) expressing Hrap did not show any symptoms of infection after artificial inoculation of potted plants in the screenhouse, whereas control nontransgenic plants showed severe symptoms resulting in complete wilting. This study demonstrates that the constitutive expression of the sweet pepper Hrap gene in banana results in enhanced resistance to BXW. We describe the development of transgenic banana varieties resistant to BXW, which will boost the arsenal available to fight this epidemic disease and save livelihoods in the Great Lakes region of East and Central Africa.