An investigation into the potential of oxalate decarboxylase enzyme, to confer tolerance to Sclerotium cepivorum in transgenic Alliums

Abstract

Allium white rot (AWR) is a major fungal disease that affects commercial Allium production. AWR is caused by a necrotrophic soil born fungus Sclerotium cepivorum Berk., which belongs to the family, Sclerotiniaceae. Oxalic acid is thought to be the major triggering component for this fungal infection on Alliums. There are no reports of satisfactory control or eradiation methods or known natural resistance against AWR within the Allium gene pool. Recent research has demonstrated that other dicots species that carry oxalate degrading enzymes exhibit resistance to sclerotial pathogens. In this study, the ability of oxalate decarboxylase (oxdc), one of the oxalate degrading enzyme to confer resistance in Allium spp against S. cepivorum was investigated. The OXDC enzyme degrades oxalate to formate and CO₂. This was exploited in transgenic Allium lines carrying two different preliminary oxdc constructs, one with Flammulina oxdc (OXDCSp-P) and the second one, Flammulina oxdc fused to sweet potato sporamine vacuole targeting signal at the N terminal (OXDCSp-Vac-P). Though transgenic Alliums produced showed the presence of functional OXDC enzyme, they failed to show any significant level of resistance to the pathogen. Moreover, there was no correlation between OXDC activity and resistance level among the transgenic Alliums tested. Vacuolar localisation of OXDC (OXDCSp-Vac-P) failed to show higher level of OXDC activity or resistance compared to transgenic plants carrying OXDCSp-P. Moreover, all transgenic plants produced with these oxdc constructs showed various degree of visible GFP fluorescence suggesting the silencing of the transgene within the transgenic lines. As part of identifying the reasons for low levels of OXDC activity, low level of resistance offered by transgenic Alliums and transgene silencing, two postulates were hypothesised. The first one was to understand the subcellular localisation of OXDC in Alliums and the second one was to design new oxdc constructs for developing another set of transgenic Alliums to address the suspected 35S mediated silencing of transgenes. Transient transformation studies were designed using the constructs that specifically targets OXDC to different subcellular localisation. In order to understand whether the preliminary constructs possessed signal peptides as it would have predicted in the Flammulina oxdc, various bioinformatics tools were used. Putative secretory signal peptide was chosen and various constructs were made to understand the subcellular localisation of the GFP fusion proteins in Alliums and tobacco. The localisation experiments showed that most of the constructs under study were localising the GFP fluorescence of fusion protein in ER. However, OXDC construct without native secretory signal peptide (OXDCΔSp::GFP) and vacuole targeting signal fused to GFP (GFPVac) localised the GFP fluorescence in Allium cytoplasm and vacuole as expected. None of the targeting constructs designed for localising OXDC in vacuole (OXDCSp-Vac::GFP and OXDCΔSp-Vac::GFP) or in apoplast (OXDCΔSp-ApoT::GFP and OXDCΔSp-ApoC::GFP) were successful. Three different constructs (OXDCΔSp-N, OXDCΔSp-Vac -N and OXDCSp-N) were designed considering the suspected 35S mediated transgene silencing in the transgenic Alliums carrying previous constructs. The OXDCΔSp-N was designed to target the protein to cytoplasm, OXDCΔSp-Vac-N was to target protein to the vacuole, though the localisation experiment showed that OXDCΔSp-Vac::GFP localised GFP fluorescence in cytoplasm. The OXDCSp-N was expected to localise OXDC in ER as per the localisation results. The qRT-PCR analysis showed the presence of oxdc transcript and the variation in the transcript abundance within the same transgenic line. However, no correlation between transcript level and OXDC activity was observed as reported by other researchers. None of the transgenic garlic lines produced carrying new OXDC constructs showed any higher levels of OXDC activity or resistance to pathogen compared to the transgenic Alliums produced by previous constructs. The results of this research study is inefficient to confirm the use oxalate decarboxylase as a suitable enzyme for the production transgenic Alliums with enhanced tolerance to S. cepivorum. This leads to suspect that Alliums may be sensitive to the presence of any OA-independent molecules that are in play during S. cepivorum infection / AWR. Further study is required using S. cepivorum mutants deficient in OA production to understand the presence of any other OA-independent elicitors. Allium signalling pathway needs to be investigated in order to achieve high localised transgene expression to fight against fungal infection.