This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. Fusarium wilt disease caused by fungal pathogen Fusarium oxysporum f. sp. cubense (FOC) is one of the major threats to the banana cultivation (Ploetz and Pegg, 2000). The fungal mycelium clogs the xylem vessels and hinders the supply of water and nutrients, showing the symptoms of discoloration and drooping of leaves, splitting of stem, eventually leading to the collapse of the plant (Li et al., 2011). Based on vir ulence a nd host specificity FOC ha s been differentiated to four physiological races viz., races 1-4 (Moore et al., 1993). Banana cv. Rasthali is highly susceptible to FOC1, due to which the area of cultivation has declined from 146 to 20 hectares (Singhal, 1999), continued cultivation in sa me condition worsen the situation which threatens total extinction (Thangavelu et al., 2001). Currently available methods to control the disease are ineffective, hence deploying resistance in susceptible variety by genetic transformation served as an alternative strategy (Ploetz, 2015). F. oxysporum species known to exhibit shor t biotr ophic pha se followed by complete necrotrophy in host plant (Thaler et al., 2004). In view of this, employing genes which negatively regulate the cell death pathway in host were used to enhance resistance against necrotrophic fungi (Dickman and de Figueir edo, 2013). BAG genes encoding multifunctional group of pr oteins function by interacting with the signaling molecules and molecular chaperones, such as heat shock proteins resulting in inhibition of PCD (Sonderma nn et al. , 2001; Doukhanina et al., 2006; Jacobs and Marnett 2009; Ge et al., 2016). Most of the time, exogenous genes have mostly been expressed by ubiquitin promoter that drives high-level expression of transgenes in monocot plants (Jiang et al., 2018). Beta-glucosidase promoter isolated from Zea mays (ZmBgl) shows much stronger activity in root parts (vigorous cell division zones associated with vascular tissues) compared to mature seeds and edible part of the crop (Gu et al., 2006). Over expression of programmed cell death (PCD) gene, AtBAG4 under two different promoters namely ZmBgl promoter and ubiquitin promoter was studied to confer FOC1 resistance in banana cv. Rasthali. The embryogenic cell suspension (ECS) initiated from male inflorescence of banana cv. Rasthali was maintained in controlled condition as previously Short Communication J. Hortic. Sci. Vol. 18(1) : 228-232, 2023 https://doi.org/10.24154/jhs.v18i1.2169 Over expression of anti-apoptotic gene in banana cv Rasthali enhances resistance against Fusarium oxysporum f. sp. cubense Race 1 Umesha M.1, Sunisha C.2, Usharani T.R.2*, Sowmya H.D.2 and Sriram S.3 1Dept of Biotechnology and Biochemistry, Centre for Post-Graduate Studies Jain University, Bengaluru - 560069, Karnataka, India 2Division of Basic Sciences, 3Division of Crop Protection ICAR-Indian Institute of Horticultural Research, Hesaraghatta, Bangalore - 560089, Karnataka, India *Corresponding author Email : Usharani.TR@icar.gov.in ABSTRACT The most popular banana cv Rasthali was transformed with anti-apoptotic gene, AtBAG4 regulated with two different promoters viz., ZmBgl and ubiquitin to enhance the tolerance levels to Fusarium oxysporum f. sp. cubense Race 1 (FOC1). The differences in gene expression driven by two promoters revealed that stronger expression of AtBAG4 gene under the ubiquitin promoter suppressed the infection and spreading processes of FOC1 in transgenic banana under standard bioassay systems. Analysis using the real time PCR showed the varying levels of AtBAG4 gene expression under two promoters. It was evident that ZmBgl driven AtBAG4 lead to lower gene expression in leaves which correlated with lesser levels of resistance to FOC1. Constitutive expression of AtBAG4 under the control of ubiquitin promoter showed increased transgene transcripts which directly correlated with the enhanced tolerance against FOC1 from seedlings stage to active vegetative phases. This study reveals the importance of constitutive expression of anti-apoptotic gene showing enhanced tolerance against the most dreaded FOC1 in highly susceptible variety Rasthali. Keywords : Anti-apoptotic gene, banana, constitutive expression, fusarium wilt, rasthali 229 J. Hortic. Sci. Vol. 18(1) : 228-232, 2023 described (Sunisha et al., 2020a). ECS was heat shocked at 45ºC for 5 min and transformed with AtBag4 gene constructs obtained from Queensland Univer s it y Tec hnology, Au s t r a lia u s ing A g ro b a c t e r i u m s t r a in AG L a s des c r ib ed b y (Khanna et al., 2004). The putative transformants developed with AtBAG4 genes driven by ZmBgl pr omot er a s well a s ubiquit in pr omoter wer e s u b jec t ed t o P C R a na lys is . Tota l R N A wa s extracted from leaf tissue of untransformed and transformed plants using plant RNA isolation kit (Sigma , Aldr ich). Br ief ly, 4 µ g of RNA wa s subjected for DNa se treatment and first strand cDNA synthesized using 2µg of RNA, Oligo (dT) 12-18 primer (Sigma), RevertAid TM M-MuLV Reverse Transcriptase (Thermo Scientific). Five transgenic lines f r om ea ch c ons t r u ct wer e individu a lly subjected to qPCR in three replications with a total volume of 20 μL reaction mixture containing 10 μL of PCR mix from SYBR Green kit (Takara), 1 μL of gene primer set and 5 μL of cDNA template in a 7 5 0 0 R e a l- Time P C R s ys t em ( Ap p lied Biosystems). Banana Actin gene was used as an endogenou s gene c ont r ol f or qRT- P C R . Amplification conditions were, initially 95 °C for 5 min followed by 40 cycles of 95 °C for 1 min, 60 °C for 45 sec, and 72 °C for 30 sec, each. Final PCR melt curve of 95°C for 1min and 60°C for 15 s was employed and 2-ΔΔCT method was used to a na lyze qua ntitative var ia tion (Sunisha et al. , 2020b). Five transgenic lines from each construct a long with the untr a nsfor med susceptib le cv. Rasthali and resistant cv. Grand Naine controls were inoculated with FOC1 obtained from Plant Fig. 1 Comparative AtBAG4 gene expression analysis in leaves of transgenic plants Lines 8, 9, 142, 166 and 168 represent transgenic banana cv Rasthali expressing AtBAG4 under ZmBgl promoter. Lines 333, 335, 339, 343 and 345 represent transgenic banana cv Rasthali expressing AtBAG4 under ubiquitin promoter pathology labor atory, ICAR-Indian Institute of Hor ticu ltur e Res ea r c h, Ba nga lor e (India ), a s described previously by Smith et al. (2008) with slight modification in the potting mixture. External s ymp t oms w er e r ec or ded f ou r weeks p os t inoculation by scoring each plant for yellowing and wilting symptoms (Paul et al., 2011) using a 1–5 point scoring, where 1 represented -healthy, no symptoms; 2—slight symptoms (yellowing of the leaves); 3—advance symptoms (dropping of the leaves); 4—extensive symptoms (whole foliage got dried); 5—entire plant affected (complete dead plant). The data was analyzed by one-way analysis of var iance (ANOVA) using GraphPad prism® software (USA). Table 1. Bioassay of AtBAG4 transgenic lines of banana cv. Rasthali plants inoculated with FOC1 for symptom expression Sl. No. Transgenic Lines External symtoms ZmBgl: Uboquitin: ZmBgl:AtBAG4 Ubiquitin:AtBAG4 AtBAG4 AtBAG4 Yellowing Wilting Yellowing Wilting 1 168 333 3.3 1.3 2.3 1.6 2 166 335 3.0 2.0 2.6 1.3 3 142 339 3.3 2.3 2.6 1.6 4 8 343 2.6 1.6 2.3 2.0 5 9 345 2.3 2.6 1.6 2.0 6 RS CONTOL 4.3 4.6 5.0 4.3 C.D. 0.9 1.2 0.8 1.1 Constitutive expression of anti-apoptotic gene in banana 230 Tr a ns genic lines ex p r es s ing At BAG 4 gene; R S— contr ol a r e u nt r a nsf or med c v R a s t ha li. Results a re presented as score—yellowing a nd wilting: 1–5 scale, stem splitting: 1–3 scale. Five to seven leaf stage plants were subjected for the FOC root-challenge bioassay. The treatments were significantly dissimilar from susceptible RS control lines as P < 0.05 based on LSD post hoc test Agrobacterium mediated transformation of ECS with binary vector containing AtBAG4 gene, driven by ZmBgl and ubiquitin promoter resulted in total of 23 PCR confirmed putative transformants free f r om s oma c lona l va r ia t ion. T he well r oot ed plantlets were acclimatized in net house for further development. The efficient overexpression a nd transcript levels of the AtBAG4 gene were examined using cDNAs derived from five selected transgenic lines of each construct and also in un-transformed cont r ol. T he qRT-P CR da t a showed t ha t the expression of AtBAG4 gene driven by ubiquitin promoter ranged from 2 to 8.5-fold whereas the transgenic lines expressing AtBAG4 gene under Z mBgl p r omot er s howed les s t ha n one- f old expression in leaves compared to untransformed plants (Fig 1). The differential expression in the t r a ns genic a nd non- t r a ns for med pla nts wer e statistically significant (F = 131.7; P = < 0.0001). Tr a nsfor ma nts expr essing AtBA G4 dr iven by ubiquitin and ZmBgl pr omoter showed reduced external symptoms compared to untransfor med control plants four weeks post inoculation. The ubiquitin promoter resulting transgenic events exhibited higher gene expression level in leaves compa red to tr ansgenic events developed with ZmBgl pr omot er. Also, tr a nsgenic lines over expressing anti-apoptotic AtBAG4 gene driven by ubiquitin promoter exhibited reduction in external symptoms (< 3 rating for yellowing and wilting) and enhanced tolerance to Fusarium wilt caused by FOC 1 under pot condition. Similarly, the ubiquitin p r omot er u sed in t r a ns genic pla nt s f or over expressing MusaBAG1 showed enhanced resistance to FOC infection (Ghag et al., 2014). It is evident from the present study that anti-apoptotic AtBAG4 gene driven by root specific ZmBgl promoter was ha ving lower levels of ex pr es sion in lea ves . Similarly, the root specific ZmRCP-1 promoter is ola t ed f r om ma iz e f a iled t o d eliver gu s A exp r es sion in lea ves of t r a nsgenic p la nt a ins ( O nya ngo e t a l . , 2 0 1 6 ) . H owev er, t he gene expression in roots and vascular discoloration index post FOC inoculation are to be further studied to know the efficacy of root specific ZmBgl promoter over constitutive ubiquitin promoter. Further, the ZmBgl promoter expressing in vascular tissues is developmentally and spatially regulated promoter and such pr omoters are under the influence of several endogenous elements (Schmitz et al., 2022) which could be the reason for lower levels of tr ansgene expr ession in leaves. In contra st the constitutively over expressing AtBAG4 gene are expressed in all the tissues leading to higher oxygen scavenging activity and preventing tissue damage restricting the faster multiplication of FOC1 (Paul et al., 2011). Owing to the multiple reports confirming host PCD manipulation induced resistance to various FOC races (Magambo et al., 2012; Dale et al., 2017), the modification of genes regulating PCD pathways in plants is emerging as a promising strategy to provide broad-spectrum resistance to both biotic and abiotic stresses (Lincoln et al., 2002; Li and Dickma n, 2004;). Hence, our r esear ch ma inly focused on improving resistance to Fusarium wilt by manipulating PCD pathways. ACKNOWLEDGEMENT T he a uthor s a r e tha nkful to DBT-BIRAC for f u nding u nder t he p r ojec t D evelop ment a nd Transfer of Technology from Queensland University of Technology (QUT ), Austr a lia to India for Biofortifcation and Disease Resistance in Banana. 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