Expression of radish defensin (RsAFP2) gene in chickpea (Cicer arietinum L.) confers resistance to Fusarium wilt disease

Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity. In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms. Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea.


Introduction
Chickpea (Cicer arietinum L.) is the third most nutritionally important pulse crop after dry beans and peas worldwide. It is cultivated worldwide and is an important dietary source of proteins for human consumption [1][2][3]. Chickpea is susceptible to several biotic and abiotic stresses [3]. Among different biotic stresses, fungal diseases like Fusarium wilt (Fusarium oxysporum f. sp. ciceri) and Ascochyta blight (Ascochyta rabiei) are the most prominent fungal diseases in tropical and temperate regions of the world [4]. The F.oxysporum f. sp. ciceri is one of the devastating soil-borne fungi of the chickpea [4]. Developing biotic and abiotic stress-resistant genotypes using conventional breeding is a time-consuming and challenging task. The biotechnological methods offer prospects for improving the chickpea with novel traits, especially resistance to different pathogens [5][6][7][8].
Plant defensins are composed of small, basic cationic peptides (with a length of 45-54 amino acids) consisting of one α-helix and three β-sheets with four disulfide bonds of cysteine-stabilized with α-helix β-sheet motif (CSα/β) [9,10]. These peptides play an essential role in defense against a wide variety of fungal pathogens [11,12]. Plant defensins interact with specific membrane components to trigger intracellular signaling cascades that obstruct pathogen growth [13]. The plant defensin peptides can inhibit pathogenic fungal growth and are non-toxic to most plant and animal cells [14]. Most plants produce different defensin molecules upon the pathogen attack and help in defending the plants from pathogens [9,10]. Plant defensins are essential candidate genes for developing transgenic plants resistant to multiple diseases caused by bacterial, fungal, and viral pathogens. Several transgenic crop plants expressing higher levels of 1 3 defensins could increase resistance to pathogens and reduce crop losses [15][16][17].

Plant material and explant preparation
Seeds of chickpea (cv. JAKI9218) were procured from Germplasm Resource Unit, ICRISAT, Pantancheru, Hyderabad, Telangana State, India. The 70% ethanol was used to sterilize seeds for 1 min, followed by HgCl 2 with 0.1% for 10 min, then rinsed five times with sterile distilled water. Surface sterilized seeds were imbibed overnight in sterile distilled water. The imbibed seeds were blotted dry, and then the seed coat was taken off and placed on MSB5 [31,32] medium fortified with 1.0 mg l −1 BAP (15 per plate) at 24 ± 2 °C in dark condition for one day. The embryo axis explants were prepared by dissecting out the cotyledons, shoot (plumule), and radicle (root apex) [33,34]. The embryo axes (EA) were pricked and pre-cultured for three days on a co-culture medium (CCM) employed for transformation experiments [35].

Agrobacterium strain, plasmid vector, and culture preparation
The present investigation used the Agrobacterium tumefaciens strain LBA4404 harboring pFAJ3006 with RsAFP2 gene in this investigation (The plasmid vector pFAJ3006 was used kindly provided by Dr. Bruno P A Cammue, Centre for Microbial and Plant Genetics, KU Leuven, Heverlee, Belgium). A single colony was inoculated in 5 ml LB fortified with rifampicin (10 mg l −1 ) and kanamycin (50 mg l −1 ). Then the LB broth was incubated at 28 °C at 200 rpm overnight in a rotary shaker. The overnight grew culture was added to 30 ml LB broth with the same concentrations of antibiotics and incubated at 28 °C with 200 rpm until the OD600 reached ~ 1.0. The bacterial pellet was obtained after centrifuging at 6000 rpm for 10 min. Subsequently, the bacterial pellet was suspended in a liquid infection medium (LIM) with pH 5.8 to get a suitable OD600 of 1.0 used to infect the explants [35].

Pre-culture and plant transformation
The embryo axis explants were pre-cultured on a co-cultivation medium (CCM) for three days. The explants were infected by immersing in the Agrobacterium suspension for 30 min with intermittent agitation and dried on a sterilized filter paper. The infected explants were inoculated on CCM augmented with acetosyringone (100 µM), l-cysteine (200 mg l −1 ), IBA (0.05 mg l −1 ), and BAP (2.0 mg l -1 ). The cultures were incubated for three days in the dark at 24 ± 2 °C in the culture room. Ten explants were placed on the solid co-cultivation medium in each petri plate (90 mm diameter × 15 mm deep). The responsive explants were subcultured for every 10-15 days. After molecular analysis, the number of transgenic plants was counted and calculated.

Selection and regeneration of transformants
The co-cultivated explants were treated with liquid MSB5 medium containing cefotaxime (200 mg l −1 ) followed by drying on sterile filter paper. The explants were inoculated onto SIM and then sub-cultured at 10-15 d intervals. They were transferred onto SIM-1 (without selection agent) and kept for 7-10 days. The responding explants were transferred onto SIM-2 supplemented with kanamycin (25 mg l −1 ; selection agent) and incubated for 10-15 days. Then the explants were inoculated onto SIM-3 supplemented with kanamycin (50 mg l −1 ) for another 10-15 days. The explants with prominent shoots were shifted, to SIM-4, amended with kanamycin (75 mg l −1 ), and incubated for 10-15 days. The explants with putative transgenic shoots were transferred on SIM-5 fortified with kanamycin (100 mg l −1 ) and subcultured for two to three subcultures to eliminate escapes and chimeric shoots.

Rooting of putative transgenic shoots and acclimatization
The putative transgenic shoots (> 3 cm) were separated from multiple shoots and inoculated on RIM (root induction medium) supplemented with cefotaxime (100 mg l −1 ) and kanamycin (50 mg l −1 ). The putative transgenic plantlets were taken out carefully from culture vessels and washed under running tap water for 5-10 min to remove the agar sticking to the roots. The plantlets were moved to plastic cups filled with sand, sterile vermiculite, and soil. The plants were covered with polythene bags with the slightest puncture, then placed in culture for 2-3 weeks, and rinsed once in two days. After three weeks, plantlets were shifted to the greenhouse under controlled conditions.

Molecular analysis of putative transgenic (T0) plants
Histochemical GUS assay was carried out in embryo axis explants [36] after three successive subcultures, matured leaves T0 transformants, and respective controls from nontransformed plants. The genomic DNA was extracted from putative transgenic (T0) leaf samples and wild-type plants using a modified CTAB protocol [37] and analyzed for the presence of transgenes using the primers sets for the nptII gene (FP: 5′-ACT GGG CAC AAC AGA CAA TC-3′ and RP:5′-TTG AGC CTG GCG AAC AGT TC-3′) and the RsAFP2 gene (FP:5′-ATG GCT AAG TTT GCT TCT ATC-3′ and RP:5′-GGG GGA TCC TTA ACA AGG GAA ATA ACA GAT ACA-3′) to amplify part of the plant selectable marker, the nptII gene, and another transgene the RsAFP2 gene using genomic DNA as a template in a thermocycler (PCR). The plasmid pFAJ3006 with nptII and RsAFP2 genes and non-transformed chickpea plant genomic DNA were also subjected to amplification. Those served as positive and negative controls in PCR analysis. The PCR mixture containing 25 µl consisted of 50 ng of plant DNA, 1X PCR master mix (Thermo Scientific, USA), and 10 pmol of forward and reverse primers. The PCR was carried out for 30 cycles as follows: one cycle at 94 °C for 5 min (for initial denaturation), 30 cycles of reactions at 94 °C for 30 s (for denaturation), 52 °C for 30 s (for annealing nptII gene), 55 °C for 30 s (for annealing RsAFP2 gene), 72 °C for 2 min (for extension), and 72 °C for 10 min for the final extension. The amplified fragments were analyzed by electrophoresis at 100 V for 45 min in a 0.8% (w/v) agarose gel containing 0.5 μg/ml ethidium bromide and were visualized using a gel documentation system (Bio-Rad, USA).

Bioassay for Fusarium wilt disease
The fungal pathogen, Fusarium oxysporum f. sp. ciceri Race1 (NCIM 1281), was obtained from the National Collection of Industrial Microorganisms (NCIM), CSIR-NCL, Pune, India. Fusarium was inoculated on PDA (potato dextrose agar) medium and sub-cultured for sporulation for two weeks. A small portion of the mycelial block from cultures was taken to determine the sporulation. The spore samples were prepared with sterile distilled water (1000 µl) was added to the surface of mycelial blocks and mixed with a pipette. Seeds obtained from T0 transformed chickpea plants were screened for Fusarium wilt assay. Five seeds from each transgenic plant line, such as T1-3, T1-4, T1-5, T1-8, and T1-11, were surface sterilized and germinated in plastic cups. After one week of growth, the conidial suspension (~ 5 ml) was added to the chickpea seedlings. The treated with fungal pathogen and control seedlings were maintained under controlled temperature and humid conditions. The response of seedlings and symptoms were observed after fourteen days of post-inoculation (dpi) with the fungal pathogen. The chickpea seedlings were identified based on an acute withering, yellowing leaves, and plant drying. The root and stem parts of transgenic and control (non-transgenic) plants were longitudinally dissected and observed the symptoms.

Statistical analysis
Five independent experiments were conducted by co-cultivating 100-120 embryo axis explants. The number of explants with putative transgenic shoots was calculated. The number of putative transgenic shoots produced roots, and the number of putative T0 transformants (PCR positive transgenes) were tabulated. The transformation efficiency was measured and tabulated for each experiment. For each experiment, the data were scored, analyzed, and presented in the table as means ± standard error.

Generation of transgenic chickpea plants
A total of five individual transformation experiments were carried out with embryo axis explants of chickpea cv. JAKI9218. After optimizing factors influencing the Agrobacterium-mediated genetic transformation in chickpea, mechanically injured embryo axis explants were pre-cultured on a co-cultivation medium (CCM) for three days followed by infection with Agrobacterium-suspension for thirty minutes and co-cultivation for three days. To induce shoot buds, the explants were cultured on SIM-1 fortified with 250 mg l −1 cefotaxime and without kanamycin and incubated for 7-10 days. After 7-10 days of culture, the explants were transferred onto SIM-2 fortified with 25 mg l −1 kanamycin (low selection pressure) and 250 mg l −1 cefotaxime after 7-10 days incubated for 1 week. After one week of selection, responding explants were shifted to SIM-3 augmented with cefotaxime (250 mg l −1 ) and kanamycin (50 mg l −1 ) and incubated for another 7-10 days. After the second selection cycle, explants with shoot buds were inoculated to SIM-4 fortified with 75 mg l −1 kanamycin and incubated for 10 days. The putatively transformed shoots were transferred to SIM-5 fortified with 100 mg 1 -1 kanamycin repeated twice with 10 days intervals to eliminate escapes and chimeras. After the final selection, putatively transformed shoots (green and healthy) were separated from the untransformed bleached and necrotic shoots. The healthy and green putatively transformed shoots were inoculated onto SEM containing cefotaxime (100 mg l −1 ) and kanamycin (50 mg l −1 ) for two passages with 7-10 days intervals. In five independent experiments, 564 embryo axis explants were co-cultivated, 59 putative transgenic shoots were produced after the final selection cycle (Table 1). Putatively transformed shoots were elongated on SEM augmented with kanamycin (50 mg l −1 ) and cefotaxime (100 mg l −1 ). Out of 59 elongated shoots, 24 shoots were developed roots to produce putative transgenic plantlets ( Table 1). The shoots (> 3 cm) were successfully rooted in RIM augmented with kanamycin (50 mg l −1 ) and IBA (2.0 mg l −1 ). The putatively transformed plantlets were hardened in earthen pots. The plantlets were masked with polyethylene cover with a minute hole and placed in the culture room for 2-3 weeks. The plantlets were shifted to earthen pots filled with sterile vermiculite, sand, and soil and grown in the greenhouse. A final of 18 plantlets was hardened under culture room conditions 12 plantlets survived successfully in field conditions.

Molecular analysis of transgenic chickpea plants
Histochemical GUS expression assay was conducted in embryo axis explants and shoot buds produced after Agrobacterium infection. The strong intensity blue colour was observed in shoots derived from embryo axis explants. No GUS activity was observed in non-infected explants (control). PCR amplification of the nptII and RsAFP2 genes using genomic DNA of 12 putative T0 transformants regenerated from embryo axis explants alongwith control (wildtype) plant and positive control (plasmid of pFAJ3006). PCR analysis of 8 putative T0 transformants regenerated from embryo axis explants (randomly chosen from 12 PCR positive plants) along with control (wild-type) plant and positive control (plasmid pFAJ3006) is shown in Fig. 1a, b. PCR amplification analysis of DNA from the putative T0 transformants derived from embryo axis explants yielded the expected 457 bp amplicon of the nptII gene and 243 bp amplicon of the RsAFP2 gene was visible by exposing the EtBr stained gel in UV light. The presence of RsAFP2 and nptII genes was confirmed in 18/24 genomic DNA samples of plants regenerated from embryo axis explants (Table 1) whereas no amplification of transgenes was observed in control (non-transformed) plants.  (T1-3, T1-4, T1-5, T1-8, and T1-11) tested, three lines (T1-3, T1-5, and T1-8) did not show any symptoms upon fusarium infection. These transgenic lines appeared healthy even after four weeks of incubation. In contrast, two transgenic plants (T1-4 and T1-11) showed a delayed period of symptoms development (after 3-4 weeks) of infection. The control plants (wild-type) exhibited severe wilt symptoms after Fusarium infection. The control plants showed the wilting and dark browning of the inner vasculature (Fig. 2a, b), causing the death of the plants at the end of the second week. The seedling of three transgenic lines (T1-3, The other transgenic (T1-4 and T1-11) seedlings showed different resistance levels. All these transformed lines maintained the antifungal activity and appeared to be morphologically normal plants.  [22]. Transgenic rice plants were developed using A. tumefaciens strain LBA 4404 harboring pUb1-Rs-T [24]. Several transgenetic plants were produced using different plasmids and Agrobacterium strains with the RsAFP2 gene.

Molecular confirmation of transformants
In the present study, GUS expression was observed in shoots and leaves from putative T0 transformants, indicating transient GUS gene expression. No background GUS activity was observed in individual control plant parts from nontransformed plants. Similar results have been reported in earlier genetic transformation studies in chickpeas [38,39]. In the present study, nptII and RsAFP2 transgenes were amplified using two gene-specific forward primers and reverse primers with genomic DNA from putative transgenic plants.
The present study confirmed no amplification of DNA in non-transformed plants. In contrast, the expected and 457 bp amplicon of the nptII gene and 243 bp amplicon of the RsAFP2 gene were observed in putative T0 transformants (Fig. 1a, b). The stable integration of the RsAFP2 gene in the genome of several transgenic plants have been reported such as tobacco [19,29], tomato [21,22], apple [20], rice [23,24], poplar [25] and peanut [28,30]. The amplification was found only in DNA from transformed chickpea plants overexpressed with the RsAFP2 gene and not in the DNA of the non-transformed plants. The PCR amplified fragments of the RsAFP2 gene showed the stable integration of the RsAFP2 gene into the chickpea genome.

Bioassay for Fusarium wilt
Transgenic chickpea plants overexpressing RsAFP2 showed enhancement in their resistance to Fusarium oxysporum f. sp. ciceri. A similar enhancement of resistance to different fungal pathogens was reported in several transgenic plants.
The expression of radish defensin (RsAFP2) in tobacco increased resistance to Alternaria longipes [18], P. parasitica var. nicotianae, A. alternata f. sp. tabaci and P. syringae pv. tabaci [19]. The RsAFP2 gene showed increased resistance to F. culmorum in transgenic apple plants [20]. The  [21], F. oxysporum f. sp. lycopersici and B. cinerea [22]. The transgenic oilseed rape plants conferred resistance to S. sclerotiorumi and V. dahlia [21]. Transgenic rice plants with the RsAFP2 gene conferred increased resistance to M. oryzae and R. solani [23,24]. The expression of fusion genes (SniOLP and RsAFP2) enhanced resistance to P. personata in transgenic peanut plants [28]. Ouyang et al. [25] developed transgenic Eucalyptus plants with the RsAFP2 gene that showed enhanced resistance to P. capsici. The fusion gene (Tfgd2-RsAFP2) conferred resistance against R. solani and P. parasitica var. nicotianae in transgenic tobacco plants [29]. Three transgenic lines (T1) seedlings showed superior tolerance against fungal infestation during the Fusarium wilt assay observations. The control (non-transgenic) seedlings treated with pathogen were affected seriously. Similarly, the two transgenic lines showed different resistance levels, which could be simultaneous with the expression level of nptII and RsAFP2 genes (Fig. 1a, b). These observations agree with the resistance levels that have been reported for the expression of BjNPR1 gene in mungbean seedlings during R. solani (root rot) infection [40]. Similarly, rice transgenic plants constitutively expressing Dm-AMP1 had a high level of resistance against sheath blight and blast diseases caused by R. solani and M. oryzae, respectively [41]. Transgenic tomato plants with MsDef1 gene exhibited different resistance levels against Fusarium wilt disease caused by F. oxysporum f. sp. lycopersi [42]. The transgenic banana plants overexpressing the Ace-AMP gene showed resistance to fusarium wilt diseases caused by wilt disease (Fusarium oxysporum f.sp. cubense) [43]. Our observations are concurrent with transgenic peanut plants constitutively expressing fusion genes (Tfgd2 and RsAFP2) exhibiting different resistance levels to early and late leaf spot diseases [29]. In this study, the transgenic approach generated transgenic chickpea plants tolerant of Fusarium wilt by expressing the radish defensin gene. These transgenic lines will be further evaluated for their resistance to other fungal diseases and may be used as additional sources of disease resistance for chickpea breeding programmes.

Conclusion
In this study, we have shown that overexpression of the RsAFP2 gene conferred improvement in resistance to Fusarium wilt disease caused by Fusarium oxysporum f. sp. ciceri in the chickpea genotype JAKI9218. The results thus demonstrate the potential use of genetic transformation technology to improve the chickpea germplasm. Besides, the regeneration protocol developed in the present investigation has been successfully produced transgenic chickpea plants to confer resistance to Fusarium wilt disease. This protocol can also be utilized to transfer agronomically functional gene(s) to improve the chickpea cultivars.