OVEREXPRESSION OF A NOVEL DUAL FUNCTION PURINE NUCLEOSIDE PHOSPHORYLASE AND AN ANTIMICROBIAL 2S ALBUMIN PROTEIN TO ENHANCE MULTIPLE STRESS RESISTANCE IN CITRUS

Abstract

The world population is projected at 8 billion as of 2023 and is estimated to exceed 10 billion by the end of 2050. In between this increasing population, fruit production has consistently been inadequate to meet the required demand. Citrus is one of the most important fruit crops which is cultivated worldwide in more than 140 countries. Citrus is known to have antioxidant, anti-inflammatory, anticancer, antimicrobial and anti-insect activities and also effective against several diseases including cardiovascular disorders, diabetes, obesity, cancer, etc. The global citrus industry is huge, 158.5 Mt of citrus fruits were produced in summer 2021 and winter 2021/22 seasons. Regardless of the global production and popularity, significant changes in the climatic/environmental conditions has negatively impacted citrus fruit crop production and quality. Citrus spp. is susceptible to both, abiotic stresses including drought, salinity, alkalinity, flooding, extreme temperatures, oxidative stress and nutrient deficiency and biotic stresses such as vectors, sap feeders, mites, bacteria, herbivores, viruses and fungi. Under natural conditions, citrus experiences several stresses simultaneously, which causes severe and irreversible damage to the fruit crop. Among all the diseases, Haunglongbing (HLB; citrus greening) is the most destructive with no permanent cure. HLB is caused by a phloem-limited, non-culturable, gram-negative, fastidious bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) which is vectored by Diaphorina citri (Asian citrus psyllid) and is one of the major factors for declining citrus production. Thus, efficient and timely solutions are needed. Several limitations of conventional breeding have prompted the scientific communities to develop efficient methods for enhancing stress resistance/crop improvement in citrus. Among all the developed method, Agrobacterium-mediated transformation of in vitro germinated epicotyl segments is the most popular however, several limitations are also present. Citrus is a non-host to Agrobacterium thus, many commercial species including Citrus aurantifolia and Citrus sinensis are recalcitrant to the transformation. In the present study, we evaluated every step of Agrobacterium-mediated transformation of C. aurantifolia and C. sinensis and included innovative and needful improvements. We provided callus-inducing treatments to the epicotyls during pre-culture and co-cultivation by supplementing auxin and cytokinin rich media. Post-transformation, we screened different combinations of hormones for both species to obtain maximum regeneration. C. aurantifolia required a combination of higher BAP and lower NAA while lower BAP alone, produced maximum numbers of shoots per epicotyl in C. sinensis. We modified the protocol of in vitro micrografting to combat loss of transgenic lines. To achieve this, the apical portion and nodes of elongated GUS positive shoots were separately in vitro micrografted on different rootstocks, making negligible loss at this step. Additionally, transgenic citrus shoots do not root, so we also developed optimal rooting condition for GUS positive shoots for both citrus species. Using this protocol, the whole transgenic plants of C. aurantifolia and C. sinensis can be developed in about ~4 months with transformation efficiency of 30% and 22 % for respective species. Further, we constitutively overexpressed PRpnp, a PNP family protein with trypsin inhibitory activity, in C. aurantifolia Cv. Pramalini and C. sinensis Cv. Mosambi which showed nuclear-cytoplasmic localization in tobacco upon transient overexpression, where it could regulate various important biological functions by maintaining intracellular purine pool. To evaluate the stress responsive role of PRpnp, we selected transgenic C. aurantifolia and exposed it to several abiotic and biotic stresses and compared to WT. Overexpression of PRpnp significantly enhanced tolerance to salt, oxidative stress, alkaline pH, drought and two pests, Papilio demoleus and Scirtothrips citri in transgenic plants. To reveal the underlying mechanism of multiple stress resistance, we performed global gene expression studies which showed up-regulation of DEGs, related to ABA- and JA- biosynthesis and signaling, plant defense, growth and development. The transcriptome results were confirmed via LC-MS/MS analysis which showed increased endogenous accumulation of ABA and JA in transgenic plants. Overall, PRpnp enhances multiple stress resistance by enhancing the endogenous ABA and JA, which interact synergistically and it also inhibits trypsin proteases in the insect gut to enhance stress tolerance in transgenic Mexican lime. Being a purine salvage enzyme, we anticipated the role of PRpnp in CK metabolism. LC-MS/MS analysis showed enhanced endogenous levels of CK-free bases in transgenic plants thus, confirmed the role of PRpnp in CK metabolism. In conclusion, we found overexpression of PRpnp enhanced overall plant vigour and enhanced multiple stress resistance in transgenic C. aurantifolia. Furthermore, to enhance HLB resistance, we overexpressed P2SA under phloem-specific promoter (AtSUC2) in C. aurantifolia and C. sinensis while it showed its localization to prevacuolar compartments (PVCs) in tobacco cells. The localization P2SA to PVCs is attributed by its nature of being a storage protein. To evaluate the antimicrobial role of P2SA against CLas, we used two approaches. In first one, we grafted transgenic citrus scions over HLB infected citrus rootstocks (WT) whereas, the second approach involved the grafting of HLB infected scions (WT) over transgenic rootstocks. After three months of grafting, the CLas population was evaluated at a regular interval of 30 d till 210 d in transgenic and infected/control parts/plants. The results showed that at every point of treatment, the CLas population was lower in the transgenic part compared to HLB infected counterpart or control plants. After successful graft union establishment, CLas was detected in the transgenic part however, the antimicrobial property of P2SA decreased its population thus, enhancing HLB resistance in transgenic citrus. Additionally, it was apparent that upon the successful establishment of phloem connections, P2SA reduced the bacterial titer also in HLB infects scions. Thus, confirming its DNase activity against CLas in vivo.