Exploring the potential of a newly discovered Arabidopsis gene as a molecular trait in banana for increasing resistance against fungal disease

Banana (Musa spp.) is one of the major crops globally, as it is an essential food source for millions of people as well as an important export product for many (sub)tropical countries. Banana production is threatened by diverse abiotic (e.g. drought, soil salinity, temperature extremes) and biotic (e.g. diseases) factors. Of relevance for this study are the two most important fungal diseases in banana; Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis. Various approaches have been used to manage these fungal diseases, including cultural measures (e.g. removing infected materials, crop rotations), chemical and biological control agent applications, and growing resistant cultivars. Banana resistant cultivars can be generated either by conventional breeding to exploit pre-existing resistant traits in wild banana or by genetic modifications using molecular traits from banana itself or other species. Conventional breeding is hampered by very low fertility or sterility of commercial banana cultivars, making genetic modifications the most viable strategy to develop resistant banana against these fungal diseases. When this study was started, a banana genome sequence had not yet been released. Therefore, we opted for identification and isolation of molecular traits against these fungal diseases in other plant species, more specifically the model plant Arabidopsis thaliana. Through screening of activation-tagging mutants, the E3 ubiquitin ligase AtSFO1 was isolated as a potential positive regulator of the sensitivity of Arabidopsis thaliana to Foc. To gain insight into the role of AtSFO1 in plant immunity, several molecular aspects of AtSFO1 have been studied, including protein structure, ubiquitination, tissue-specific expression, subcellular localization, and expression analysis upon treatment with defense-related phytohormones In addition, we identified the banana ortholog of AtSFO1, namely MusaSFO1. Expression analysis of MusaSFO1 in banana plants infected with Mycosphaerella fijiensis showed that MusaSFO1 expression was induced in the resistant cultivar ‘Tuu Gia’ and down-regulated in the susceptible cultivar ‘Williams’. Therefore, transgenic MusaSFO1-overexpressing banana plants were generated and subjected to disease assays with Mycosphaerella fijiensis using a leaf disk bioassay. Since AtSFO1 has E3 Ub ligase activity, we further investigated which proteins in Arabidopsis are targeted for degradation. LSU1 (response to Low Sulfur 1), a protein with an unknown function and 14-3-3κ, a regulator of various biological processes in plants, were identified as interaction partners of AtSFO1 through a yeast-two-hybrid screening. Using a transient Arabidopsis leaf mesophyll protoplast system, in vivo interactions of AtSFO1-LSU1 and AtSFO1-14-3-3κ could be confirmed. However, up to now it remains uncertain whether LSU1 and 14-3-3κ are direct targets of AtSFO1 and additional experiments are needed. After further characterization of LSU1 and 14-3-3κ, including protein structure, tissue-specific expression and subcellular localization, overexpression plants of LSU1 and 14-3-3κ were challenged with Foc tropical race 4 to investigate their role in the defense response of Arabidopsis. Based on these data, a role for LSU1 and 14-3-3κ as negative regulators of defense response against Foc TR4 in Arabidopsis thaliana could be suggested. A next approach of generating transgenic banana against fungal diseases is to exploit banana endogenous molecular traits, particularly with the release of the banana sequenced genome. To study gene function, a gene silencing strategy (e.g. RNAi-induced gene silencing) is necessary. However, RNAi-induced gene silencing has not been demonstrated in banana. Therefore, using embryogenic cell suspension transformed with β-glucuronidase (GUS) as a model system, we assessed silencing of gusAINT using different intron-spliced hairpin RNA constructs. The results demonstrated that RNAi-induced gene silencing works in banana. In the future, the RNAi system can be applied to silence banana gene(s) for studying their functions in banana.

Publication year:2015

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