Enhanced Resistance To Fungal Pathogens in Transgenic Peanut (Arachis Hypogaea L.) Cultivar L14 by Overexpression of Gene encoding Chitinase 42 kDa from Trichoderma Asperellum SH16

This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specic Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the �eld of transgenic crops against phytopathogenic fungi.


Introduction
Chitinases (EC 3.2.2.14) are enzymes that break down the linear polymer of N-acetyl-D-glucosamine monomers known as chitin (Wang and Yang 2007).Chitinases may be found in a wide range of organisms, including bacteria, fungus, animals, and plants ( Peanut (Arachis hypogaea L.), which belongs to the family Fabaceae (or Leguminosae), is a high-value grain legume crop derived from Central Brazil and widely cultivated in tropical and subtropical regions for its edible oil and seeds (Pal et al. 2014;Singh et al. 2021).It is, however, one of the crops sensitive to serious diseases like stem rot, root rot, and pod rot caused by a variety of soilborne pathogens such Rhizoctonia solani, Aspergillus niger, and Sclerotium rolfsii (Ismail et al. 2007; Gour et al. 2012;Thiessen et al. 2012;Xu et al. 2015).
The method of using Agrobacterium tumefaciens to transfer a gene of interest into plant cells, resulting in transgenic plants, is known as Agrobacterium-mediated transformation (Gelvin 2003).Because of the advantages of transferring pieces of DNA with de ned ends and minimal rearrangement, transferring relatively large segments of DNA, integrating small numbers of copies of genes into plant chromosomes, and the high quality and fertility of transgenic plants, Agrobacterium-mediated transformation has become the most widely used method for transferring genes into plants (De La Riva et al. 1998; Pratiwi and Surya 2020).
Even though certain chitinase genes from Trichoderma or other species, such as rice and tobacco, have been incorporated into various crops to aid them against fungal infections (Nishizawa et al. 1999; As a consequence, the present study might be the rst to employ Agrobacterium-mediated transformation to transfer the chitinase gene from Trichoderma to peanuts in order to increase their antifungal activity.Different chitinase genes were employed in this study, including one wild-type gene, Chi42, from T. asperellum SH16, which encodes chitinase 42 kDa, and two synthetic genes (syncodChi42-1 and syncodChi42-2) generated from the Chi42 gene by optimizing codon usage for plant expression (Luong et al. 2021).The goal of this study is to identify high levels of expression of the two synthetic genes regulated by the tissue-speci c promoter (pAsy) in peanut roots, as well as their antifungal e cacy.As a result, peanuts with the T. asperellum SH16 42 kDa chitinase gene may be resistant to S. rolfsii phytopathogenic fungus.

Plant materials
Peanut (A. hypogaea L.) cultivar L14 from the Vietnam Academy of Agricultural Sciences' Field Crops Research Institute was utilized in this study (Hoa et al. 2021a).The seed coat was removed after 1 min soaks in 70% EtOH and 10 min sterilization with 65% NaClO/100 µL Tween 20 solution.The embryonal and de-embryonal cotyledons were separated from the seeds longitudinally for use as explants in Agrobacterium transformation.

Plant expression binary vector
In the present work, the Agrobacterium tumefaciens strain LBA4404 was employed, which contained the pNHL20 vector harboring the chitinase genes (Chi42, syncodChi42-1, and syncodChi42-2) expressing 42 kDa chitinase (Tue et al. 2021).Chi42 (HM191683.1) is a wild-type gene from T. asperellum SH16 (Loc et al. 2011).Both syncodChi42-1 (MT083802.1)and syncodChi42-2 (MT083803.1)are synthetic genes derived from the Chi42 gene optimized for codon usage for plant expression (Luong et al. 2021).Chitinase genes were driven by the root-speci c Asy promoter from peanut (Geng et al. 2014).A signal peptide of amylase 3D gene from rice (Jung et al. 2016) was situated at the 5' end of the genes guides newly synthesized chitinase to the periplasm for extracellular secretion (Fig 1).Brie y, the COOL program was used to optimize the chi42 gene for plant expression (Chin et al. 2014).The optimization algorithm included maximizing the codon context, ignoring individual codon usage, destabilizing the mRNA secondary structure at 5' termini, and limiting the presence of restriction enzyme sequences.Two sequences with high codon context scores (named syncodChi42-1 and syncodChi42-2) were randomly selected for synthesis (PHUSA Biochem Co).

Agrobacterium transformation
The Agrobacterium transformation procedure was followed as reported in our previous study (Hoa et al. 2021b).Brie y, explants were infected for 20 min with A. tumefaciens LBA4404 carrying the pNHL20 vector, then co-cultured on TDT medium supplemented with 66.6 µM BAP for de-embryonal cotyledon or 66.6 µM BAP and 9.1 µM 2,4-D for embryonal cotyledon at 25±2°C for 3 days in the dark.TDT medium was made up of 200 µg/mL acetosyringone, 3% sucrose, 0.8% agar, and MS basal medium (Murashige and Skoog 1962).After co-culture, explants were subcultured on the same medium with 100 mg/L kanamycin and 250 mg/L cefotaxime for screening transformants.Shoots were isolated from the screening culture to multiply on MS medium supplemented with 3% sucrose, 0.8% agar, 8.9 µM BAP, 0.6 µM IAA, and 100 mg/L kanamycin.Finally, single shoots from the shoot cluster were grown into whole plants on MS medium with 5.4 µM NAA and no antibiotics.Except for Agrobacterium treatment, all in vitro cultures were maintained at 25±2°C for 4 weeks at a light intensity of 2000-3000 lux and 16 h of daylight.

PCR ampli cation
The CTAB method was used to extract genomic DNA from in vitro transgenic peanut leaves, as reported by Clarke (2019).PCR ampli cation was carried out using speci c primers for DNA segments that served as chitinase gene indicators (Table 1).The reaction is made up of 20 ng of genomic DNA as a template, 10 pmol of each primer, 1 µL of Master Mix (Thermo Scienti c), and water added to a nal volume of 12 µL.The PCR settings were as follows: 15 min of genomic denaturation at 95°C, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by a nal extension at 72°C for 10 min.

SDS-PAGE and dot blot analysis
The TSP from in vitro transgenic peanut roots was extracted with phosphate buffer (pH 7) and quanti ed using Bradford's assay (1976).50 µg TSP was denatured for 10 min at 95°C before fractionation by SDS-PAGE.After that, Coomassie blue was used to stain the gel.For dot blot analysis, an equivalent amount of TSP was loaded onto a nitrocellulose membrane (Novex TM -Thermo Fisher Scienti c).The non-speci c binding on blot was blocked by 5% skim milk (Sigma-Aldrich).The primary antibody was a mouse anti-Ta-CHI42 polyclonal antibody diluted 1:2000 in TBST, Ta-CHI42 is recombinant chitinase 42 kDa derived from fungus T. asperellum SH16 (Luong et al. 2021).The secondary antibody was a 1:5000 dilution of goat anti-mouse IgG antibody conjugated with alkaline phosphatase (AbD Serotec-currently Bio-Rad Antibodies).The dot signals on the blot were developed with BCIP/NBT solution (Sigma-Aldrich, Cat No B6404) for 10 min in the dark.

Chitinase assay
The plate assay method was used to preliminarily evaluate the chitinolytic activity of plant chitinase.The pre-punched holes on the assay plate containing 1.5% agar and 1.2% colloidal chitin were lled with 50 µg TSP (crude chitinase).Colloidal chitin was made according to Murthy and Bleakley's method (2012).The assay plate was incubated at 4°C for 8 h for diffusing enzyme, and then at 28°C for 6 h for chitinolysis.The plate was colored with 0.1% Lugol's solution after incubation to detect chitin degradation (Calissendorff and Falhammar 2017).10 U/mL of bacterial puri ed chitinase 42 kDa was used as a positive control.50 µg TSP of the extract from the non-transgenic root was used as a negative control.
The chitinase activity was determined by measuring the absorbance of p-nitrophenol at 420 nm (Tsujibo et al. 1998).A reaction mixture containing 50 µg TSP and 15 µL of 2.5 mM pNpGlcNAc (Merck) as substrate was incubated at 45°C for 10 min.After that, the hydrolysis was stopped using 1 mM of 0.2 M sodium carbonate.The amount of chitinase required to release 1 µmol of p-nitrophenol from pNpGlcNAc per minute was de ned as one activity unit.A p-nitrophenol standard purchased from Merck was used to make the calibration curve.The speci c activity (U/mg protein) of an enzyme is determined by dividing its total activity (U/mL) by the TSP content (mg/mL).

In vitro antifungal activity
In vitro roots of chitinase transgenic peanuts were transferred in 1/2 PDA medium containing 10 4 S. rolfsii spore for evaluating their antifungal activity based on mycelium growth inhibition.Incubation was performed at 28°C for 96 h.20 µL (10 U/mL) of bacterial puri ed chitinase 42 kDa was used as a positive control, sterile distilled water and non-transgenic peanut roots were used as negative controls.

Statistics
Experiments were carried out with the tri-replicate.The data were given as the means and the ndings were evaluated using one-way ANOVA with Duncan's test at p = 0.05.
In a recent study, Iqbal et al (2012) obtained 70% of surviving plantlets on the selective medium from cotyledonary nodes of peanut and 40% of which were putatively chitinase-3 transgenic plants.Then, Prasad et al (2013) also obtained a total of 65 regenerated shoots from peanut mature cotyledons which were transferred rice Rchit gene encoding chitinase.While Sharma and Anjaiah (2000) achieved a signi cant percentage (55%) of transgenic peanuts cultivar JL-24 from de-embryonal cotyledons through Agrobacterium transformation.These data seem to suggest that genotype and explant type had an impact on transformation e ciency.Although the ratio of shoot regeneration on the selective medium is important, the number of transgenic plants and their gene expression level are the determining factors.Of fact, Agrobacterium-mediated transformation is possible with other types of peanut explants.Iqbal et al (2012) obtained an e ciency of 42% for cotyledonary node explants.Whereas it is around 63 and 62% for embryo cotyledons or around 72 and 77% for mesocotyl-derived explants of two peanut cultivars Huayu 20 and 26 (Chen et al. 2015).

Expression of chitinase 42 kDa
The expression of three genes encoding chitinase 42 kDa in transgenic peanuts was examined using SDS-PAGE and dot blot analysis.Figure 4 illustrates several protein bands with a molecular weight of 42 kDa, as expected for T. asperellum SH16 chitinase, from various samples on the polyacrylamide gel.Transgenic peanut lines with putative recombinant chitinase were chosen for dot blot analysis.Dot signals were observed in the majority tested transgenic peanut lines and positive control, except for the non-transgenic negative control and a few others (Fig. 5).In this work, the gene syncodChi42-2 (line S2A-8) still has the strongest dot signal like our earlier study employing the 35S promoter to drive the expression of the chitinase gene in peanuts (Hoa et al. 2021).These ndings imply that chitinase 42 kDa genes were successfully expressed in transgenic peanuts.However, some transgenic individuals were unable to produce or only produced a small amount of chitinase 42 kDa due to the position effect, resulting in no or weak signals on the blots (Betts et al. 2019, Pérez-González and Caro 2019).To our knowledge, until now no studies have employed antigen-antibody interaction, such as dot blot, to determine chitinase gene expression in transgenic peanuts.The majority of studies have focussed on transcriptional expression or chitinase activity in transgenic peanuts, as well as their antifungal effectiveness (

Chitinase activity assay
Colloidal chitin hydrolysis activity of chitinase 42 kDa from transgenic peanuts was determined using the agar plate assay.The transgenic peanut lines with the highest chitinase expression are shown in Figure 6.
The largest D-d difference (about 2.5 cm) was found in syncodChi42-2 transgenic peanuts (line S2A-8), which was 1.3-and 1.7-fold greater than syncodChi42-1 (line S1A-9) and Chi42 (line WTA-2) transgenic peanuts, respectively.Where D denotes the clear zone diameter and d denotes the diameter of the prepunched hole for loading enzyme.Whereas in the non-transgenic control, hydrolysis was negligible (Fig. 6).These ndings suggest that chitinase 42 kDa was present in transgenic peanuts in an active form.
Line S2A-8 also displayed the highest chitinase speci c activity of 1124 U/mg among transgenic peanuts regenerated from embryonal cotyledon, 1.25 and 2.21 times higher than line S1A-9 and line WTA-2, respectively.However, their total activity revealed the opposite result, with the line S1A-9 (140 U/mL) being approximately 1.4 and 1.6 times higher than the lines S2A-8 (100 U/mL) and WTA-2 (87 U/mL), respectively.The non-transgenic control had the lowest chitinase activity, only 53.8 U/mg (31.03 U/mL).Overall, the chitinase expression levels in the transgenic peanuts and the tested genes were different.In every case, the synthetic genes syncodChi42-1 and syncodChi42-2 were expressed stronger than the wild-type Chi42 gene.Plants do not or only produce minimal quantities of chitinase unless they are triggered by a fungal disease, according to several studies (Grover 2012;Collinge et al. 1993;Punja and Zhang 1993).In the present study, the root extract from in vitro non-transgenic and chitinase transgenic peanuts without fungal disease infection was used to determine chitinolytic activity on the colloidal chitin plate and chitinase activity.Although non-transgenic peanuts also produced chitinase, it was only in negligible amounts, so the chitinase activity was low and the hydrolysis region was small.Peanuts with the syncodChi42-2 gene driven by the 35S promoter also exhibited the higher chitinase activity compared to syncodChi42-1 and Chi42, according to a recent study (Hoa et al. 2021).They peaked at 823 U/mg, which is 1.4 times lower than the gene regulated by the Asy promoter in this study.This demonstrates that the methods employed to optimize codon use for plant expression in previous work (Luong et al. 2021), as well as the control of genes by the root-speci c promoter in this study, were successful in peanuts.
Prasad et al (2012) and Iqbal et al (2012) improved the chitinase activity of peanuts up to 2-14 times and 1.8-6.5 times higher than non-transgenic controls, respectively, by transferring the Rchit and chitinase genes from rice.Recently, Tien et al (2021) also reported that transient expression of syncodChi42-1 and syncodChi42-2 synthetic genes in Nicotiana benthamiana was 1.7 and 2.6 times greater than wild-type Chi42 gene, respectively.

In vitro antifungal activity of chitinase
To assess antifungal effectiveness, WTA-2, S1A-9, and S2A-8 transgenic peanut lines with the highest chitinase activity were chosen.After 96 h of treatment, the transgenic roots of all three peanut lines completely inhibited the growth of S. rolfsii, which causes white mold wilt disease.The positive control also showed similar ndings.While S. rolfsii grew strongly in treatments for negative controls (Fig. 8).This investigation revealed that chitinase had signi cant antifungal activity against S. rolfsii in all three transgenic peanut lines examined, with no differences found.Our results also demonstrated that the signal peptide of the rice amylase 3D gene was effectively active in chitinase transgenic peanut roots.
Peanut recombinant chitinase was extracellularly secreted against S. rolfsii.Comparing Figures 8B, it seems that non-transgenic peanut chitinase (Fig. 8C) was also produced but not enough to completely inhibit the fungus growth.
According to studies conducted by Rohini et al (2001) and Iqbal et al (2012), chitinase transgenic peanuts outperformed non-transgenic controls in terms of resistance to Cercospora arachidicola, an ascomycete that causes early leaf spots in peanuts.Prasad et al (2012) discovered that only 0-10% of Ritch transgenic peanut seeds were infected with Aspergillus avus under in vitro seed inoculation tests.

Conclusion
The chitinase 42 kDa genes from T. asperellum SH16, which are driven by the root-speci c Asy promoter, were successfully heterologously expressed in peanut cultivar L14.All three genes, one wild-type (Chi42) and two synthetic genes (syncodChi42-1 and syncodChi42-2) were secreted extracellularly by the guidance of a signal peptide of the rice amylase 3D gene.Chitinase activity of two synthetic genes outperformed the wild-type gene but all of them exhibited a strong antifungal activity for S. rolfsii.These ndings suggest that the Chi42-derived optimized chitinase genes might aid peanuts to ght the phytopathogenic fungus S. rolfsii.
Table 2 shows that after 4 weeks of culture, 303 surviving shoots were obtained from two types of transformed explants on a selective medium containing kanamycin and cefotaxime (Fig. 2).
In embryonal cotyledons, it was just 48.5% for Chi42, 52.7% for syncodChi42-1, and 40% for syncodChi42-2, whereas in de-embryonal cotyledons, it was 54.5% for Chi42, 61.1% for syncodChi42-1, and 55.6% for syncodChi42-2 (Fig. 3).Transgenic peanut lines with putative recombinant chitinase were chosen for dot blot analysis.Dot signals were observed in the majority tested transgenic peanut lines and positive control, except for the non-transgenic negative control and a few others (Fig. 5).
Colloidal chitin hydrolysis activity of chitinase 42 kDa from transgenic peanuts was determined using the agar plate assay.The transgenic peanut lines with the highest chitinase expression are shown in Figure 6.The largest D-d difference (about 2.5 cm) was found in syncodChi42-2 transgenic peanuts (line S2A-8), which was 1.3-and 1.7-fold greater than syncodChi42-1 (line S1A-9) and Chi42 (line WTA-2) transgenic peanuts, respectively.Where D denotes the clear zone diameter and d denotes the diameter of the prepunched hole for loading enzyme.Whereas in the non-transgenic control, hydrolysis was negligible (Fig. 7).
The positive control also showed similar ndings.While S. rolfsii grew strongly in treatments for negative controls (Fig. 8).This investigation revealed that chitinase had signi cant antifungal activity against S. rolfsii in all three transgenic peanut lines examined, with no differences found.Our results also demonstrated that the signal peptide of the rice amylase 3D gene was effectively active in chitinase transgenic peanut roots.Peanut recombinant chitinase was extracellularly secreted against S. rolfsii.
Comparing Figures 8B, it seems that non-transgenic peanut chitinase (Fig. 8C) was also produced but not enough to completely inhibit the fungus growth.

Figure 1 A
Figure 1

Figure 4
Figure 4 illustrates several protein bands with a molecular weight of 42 kDa, as expected for T. asperellum SH16 chitinase, from various samples on the polyacrylamide gel.

Table 1
(Jung et al. 2016equence of speci c primers for indicators of chitinase genes.TGGTACTATGCAGCTTGACCT sequence of all three forward primers was located in signal peptide segment of rice amylase 3D gene(Jung et al. 2016) that anked 5' end of chitinase genes (data not shown).The signal peptide was used for the extracellular expression of the enzyme chitinase.

Table 2
Chitinase transgenic e ciency in peanuts through embryonal and de-embryonal cotyledon.