Agrobacterium -Mediated Transformation of pMDC140 Plasmid Containing the Wheatwin2 Gene into the Tadong Rice Genome

: Blast disease resulting from Magnaporthe oryzae fungal infection reduces annual rice yield by up to 30% globally. The wheatwin2 ( wwin2 ) is a pathogenesis-related (PR) gene that encodes for a PR-4 protein with chitinase properties that is capable of degrading chitin, a major constituent of certain fungal cell walls. However, the potential for wwin2 to contribute to M. oryzae resistance in rice is unclear. This study reports the construction of a pMDC140 vector carrying the wwin2 gene and its Agrobacterium -mediated transformation into the Tadong rice genome. In brief, the wwin2 gene was synthesized and integrated into a pMDC140 vector using Gateway cloning technology and was transformed into the Tadong rice genome. Our results show a promising high transformation rate, with more than 90% of the transformed rice calli expressing β -glucuronidase ( GUS ), the reporter gene marker. The expression of the wwin2 gene in transformed rice calli was further conﬁrmed using quantitative real-time polymerase chain reaction. In conclusion, a pMDC140- wwin2 vector was constructed, which had a high transformation rate and could consistently induce expression of the GUS and wwin2 genes in Tadong rice. Data of this study is beneﬁcial for subsequent in vitro and M. oryzae -infected ﬁeld experiments to conﬁrm the defense mechanism of the wwin2 gene towards blast disease in rice. using RT-PCR and the expressions of wwin2 and hptII genes were validated in the transformed Tadong rice calli using qPCR. The results from this study are beneﬁcial for the design of M. oryzae -infected ﬁeld experiments to conﬁrm the defense mechanism of the wwin2 gene towards blast disease in rice.


Introduction
Rice is one of the most important agricultural crops that feeds more than half of the world's population. Rice is presented with various genetic diversities, morphologies, disease resistance, and yields depending on the adaption to geographical environments and domestication [1][2][3][4][5]. Asia is the largest rice-producing region (90.6%), followed by the Americas (5.2%), Africa (3.5%), Europe (0.6%), and Oceania (0.1%) [6]. It is estimated that about 480 million tons of rice are produced yearly, with over 85% for human consumption [7]. Therefore, a steady increase in rice production is undoubtedly crucial to keep up with rice demand from population growth. However, the productivity of rice is always challenging as it is influenced by pests and diseases caused by microorganisms.
Blast is a common rice disease caused by the fungal pathogen Magnaporthe oryzae infections. This fungus is highly adapted in rain-fed upland, irrigated lowland, and deepwater rice fields [8]. The M. oryzae genome displays a high capacity to manipulate its protein degradation and amino acid metabolism to facilitate infection of a host plant and simultaneously employs several backup systems, including compensatory processes and functional redundancy, to defend against the formation of appressorium from being destroyed [9]. Blast disease is responsible for about 30% of rice production losses worldwide, which consequently reduces consumer welfare and food security [10]. A recent study to molecularly identify the presence of the pathogenesis-related (PR) genes among the traditional rice varieties in the Malaysian Borneo for blast resistance revealed that all the studied rice varieties were inherited together with the rice chitinase PR gene, but the wheatwin2 (wwin2) PR gene was absent in their genomes [11], indicating that rice adopts different tactics to fight blast disease.
Several strategies have been adopted to combat blast diseases in rice. These have included chemical control, cultural practices, and nutrition management [12]. Breeders have also searched for rice varieties that had developed disease resistance over generations. However, these strategies did not provide effective protection due to the variability of blast fungus. The pathogens can adapt quickly to overcome resistance, thus causing the crops to remain vulnerable [12]. More dejectedly, breeding of disease-resistant varieties through the conventional method is technically difficult and time-consuming. Hence, an alternative approach is through the application of recombinant DNA technology, where a foreign gene that confers the resistance traits is inserted into a rice genome.
wwin2 is a gene that encodes a class 4 PR protein to protect the plant from pathogens' invasion and activates an induced defensive mechanism in the plant [13]. The wwin2 protein was initially identified and purified from wheat kernel and was reported to exhibit strong antimicrobial properties towards a wide range of pathogens, including wheatspecific pathogenic fungi [14]. The wwin2 protein inhibits the synthesis of chitin, which is one of the major elements in the pathogenic fungal cell wall [15]. Tobacco plants overexpressing this protein were reported to be significantly more resistant to a soil-borne fungal, Phytophthora nicotianae, but it is unclear for M. oryzae which is causing the blast disease in rice [16]. The wwin2 protein elucidates a great potential towards blast resistance. Therefore, this study aims to construct a plasmid containing the wwin2 gene and transform it into the genome of Tadong (an upland traditional rice variety in Malaysian Borneo) via an Agrobacterium-mediated approach. This is the first study to transform the wwin2 gene into an upland traditional rice variety and the results from this study are useful for future evaluations of blast resistance in rice expressing the wwin2 protein.

Synthesis of wwin2 Gene and Integration into a pMDC140 Vector
The wwin2 gene coding sequence was obtained from the NCBI database (accession ID: AJ006099). The 692 bp coding sequence of the wwin2 gene between attL1 and attL2 was synthesized (Supplementary Figure S1). The synthesized gene sequence was initially cloned into a pUC57 vector as an entry clone (GenScript Biotech, Piscataway, USA). The pMDC140 destination vector containing the attR recognition sites was purchased from the Arabidopsis Biological Resource Center, Ohio State. The Gateway TM LR Clonase TM II enzyme (Thermo Fisher Scientific, Waltham, USA) that catalyzes the in vitro recombination between an entry vector (pUC57 containing the wwin2 gene flanked by attL sites) and a destination vector (pMDC140 carrying the attR sites) was utilized to generate an expression clone (pMDC140 carrying the wwin2 gene) according to the manufacturer's recommendations. The integration of the wwin2 gene into the pMDC140 was confirmed using direct sequencing.

Transformation of pMDC140-wwin2 into Agrobacterium tumefaciens
The pMDC140-wwin2 was transformed into A. tumefaciens strain LBA4404 using a protocol as previously described with modifications [17]. In brief, 1 µg of pMDC140-wwin2 was mixed with 0.1 mL of competent A. tumefaciens (~10 6 cfu/mL) in a microcentrifuge tube and snap-frozen using liquid nitrogen. The mixture was thawed in a 37 • C water bath for 5 min, and about 500 µL of Luria-Bertani (LB) broth was added to the mixture, followed by incubating at 28 • C for 2-4 h with gentle shaking. The transformed A. tumefaciens was then spread on an LB plate supplemented with 50 µg/mL of kanamycin and 34 µg/mL of rifampicin and incubated at 28 • C for 2-3 days. Positive colonies were re-cultured in liquid LB broth supplemented with 50 µg/mL of kanamycin and 34 µg/mL of rifampicin until the OD reading was between 0.8 and 1.0, and the Agrobacterium cells were pelleted for rice calli transformation.

Agrobacterium-Mediated Rice Calli Transformation
Three-week-old rice calli were used as explants for transformation. They were derived from seeds of Tadong rice variety collected from Nabalu Town, Ranau, Malaysian Borneo. Calli were pre-cultured on Murashige and Skoog (MS) medium for 72 h in dark conditions and later immersed in 100 mL of MS re-suspension broth containing the activated A. tumefaciens pellet and 200 µM of acetosyringone for 30 min, with continuous shaking at 80 rpm. The calli were blot-dried and subsequently cultured on MS co-cultivation medium and incubated in dark conditions for 3 days. One of the transformed calli was subjected to scanning electron microscopy (SEM) imaging using the procedures as previously described [18]. In brief, the callus was soaked in 2% (v/v) glutaraldehyde (diluted in 0.1 M of phosphate buffer, pH 7.2) for 24 h at 4 • C, then washed three times with 0.1 M of phosphate buffer (pH 7.2) for 10 min. After that, the callus was dehydrated via a series of graded ethanol (35%, 50%, 70%, 75%, 95%, and 100%), with each concentration for 2 cycles × 10 min. The callus was then subjected to a CO 2 critical-point drying system (Leica, model: EM CPD 3000) and later coated with gold using an ion sputtering system (EmiTech, model: K550X) before observing under a SEM (Carl Zeiss, model: MA10). After the co-cultivation periods, the calli were washed using 300 mg/L of cefotaxime and transferred to MS resting medium for 3 days. Survived calli were later selected for histochemical β-glucuronidase (GUS) assay.

Histochemical GUS Assay of the Transformed Calli
Transformed calli were soaked with 0.5 mL of GUS buffer (containing 1.0 mM of K 3 Fe(CN) 6 , 1.0 mM of K 4 Fe(CN) 6 , 50.0 mM of phosphate buffer (pH 7.2), 0.1% (v/v) of Triton-X 100, and 2.0 mM of X-Gluc) and incubated at 37 • C for up to 48 h. After that, the calli were immersed in 95% (v/v) ethanol for 24 h to remove the chlorophyll. Calli were observed under a stereomicroscope, and the intensity of the blue spot was scored according to recommendations [19]. Explant with more than 25% of blue spot coverage was recorded as transient GUS-positive. A non-transformed callus was used as a control.

Validation of Transformation Using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from transformed calli showing GUS-positive and nontransformed callus as control using the Total RNA Purification Plus Kit (Norgen Biotek Corp., Thorold, Canada) according to the manufacturer's instructions. Approximately 1 µg of the isolated total RNA was reverse-transcribed into cDNA using the Maxima TM H Minus cDNA Synthesis Master Mix with DNase Kit following the manufacturer's recommendations.
The presence of wwin2, hygromycin phosphotransferase II (hptII), and sucrose phosphate synthase (sps) genes were validated using RT-PCR. In brief, a master mix containing 100 ng of cDNA as template, 1X Taq Master Mix (Vivantis Technologies, Shah Alam, Malaysia), 3.0 mM of MgCl 2 , and 0.5 µM of primer sets targeting the genes (Table 1) was prepared with a final volume of 20 µL. The mixture was subjected to an initial denaturation at 94 • C for 2 min, followed by 35 cycles of denaturation at 94 • C for 2 s, annealing at 60 • C for 30 s, and elongation at 72 • C for 30 s, and a final elongation step at 72 • C for 5 min. The amplified products were electrophoresed in 3% agarose gel stained with ethidium bromide. The expressions of the wwin2 and hptII genes were analyzed using qPCR, while the sps gene was used as an endogenous reference. qPCR was performed using the StepOnePlus TM Real-Time PCR System (Applied Biosystems, Foster City, USA) with the SYBR-Green approach (Thermo Fisher Scientific, Waltham, USA) in a final volume of 20 µL. The qPCR reaction mixture contained 100 ng of cDNA as template, 1× ViPrimePLUS Taq qPCR Green Master Mix I with ROX (Vivantis Technologies, Shah Alam, Malaysia), and 0.5 µM of forward and reverse primers that specifically target the genes of interest ( Table 1). The amplification conditions were set at: activation at 95 • C for 2 min, followed by 40 cycles of denaturation at 95 • C for 15 s, and annealing/elongation at 60 • C for 1 min. The expression data were analyzed using StepOnePlus TM Software v2.3 (Applied Biosystems, Foster City, USA). Each sample was performed in triplicate, and the mean threshold cycle (C T ) value was calculated.

Mapping of the Newly Constructed pMDC140 T-DNA Region
The integration of the wwin2 gene into the pMDC140 T-DNA region was validated using direct sequencing (Supplementary Figure S2). The wwin2 gene was located downstream of the constitutive 2× 35S cauliflower mosaic virus promoter and upstream of the GUS reporter, followed by a nopaline synthase terminator (nos T) and the hptII gene ( Figure 1). These genes were flanked by left and right borders within the T-DNA region of the pMDC140.

SEM Imaging on Agrobacterium-Transformed Rice Callus Surface
SEM imaging showed a clear fibril material formation produced by Agrobacterium on the surface of rice callus (Figure 2a). A formation of the mucilaginous layer resulting from an unsystematic proliferation in the growing callus that created a hole and exposed the inner cell to the outer environment was also clearly seen on the surface of the callus (Figure 2b).

RT-PCR to Confirm the Transformation in Rice Calli
The concentration of the isolated total RNA from the rice calli ranged between 1039 and 2353 ng/µL, whereas the A 260 /A 280 ratio ranged between 2.000 and 2.061 (Supplementary Figure S3). The transformation of these calli was further validated using RT-PCR. Figure 4 shows that the wwin2 and hptII genes were only presented in the transformed rice calli and pMDC140-wwin2 plasmid, and not in the non-transformed callus. For the sps gene, it was not detected when using the pMDC140-wwin2 plasmid as a template but was positive in both transformed and non-transformed rice calli.

Expressions of wwin2 and hptII Genes in the Transformed Calli with sps Gene as an Endogenous Reference
The expressions of the wwin2 (mean C T value = 29.79 ± 0.36 in transformed callus 1 and 27.94 ± 0.29 in transformed callus 2) and hptII (mean C T value = 27.64 ± 0.26 in transformed callus 1 and 27.15 ± 0.96 in transformed callus 2) genes were detected in transformed calli, and C T values were undetermined in non-transformed callus for both genes ( Table 2 and Supplementary Figure S4). In addition, expressions of the sps gene as an endogenous reference were detected in both transformed and non-transformed calli.

Discussion
In this study, we constructed an expression vector (pMDC140) carrying the wwin2 gene. pMDC140 was selected since it was transformed into A. tumefaciens with high efficiency and was widely used in transgenic plant studies, including in tobacco, cottonwood, and thale cress [22][23][24][25]. One of the features of this plasmid is carrying two antibiotic selectable markers: a kanamycin resistance gene for A. tumefaciens selection after plasmid transformation and a hptII gene for transgenic plants selection after A. tumefaciens transformation, assuring the selection workflow in selecting transformed A. tumefaciens and transgenic plants. This plasmid also contains the attR sites, which subsequently allow rapid and precise integration of the genes of interest into the plasmid T-DNA region by utilizing the Gateway ® technology and eliminating the use of restriction endonucleases and ligases in the entire cloning process.
A. tumefaciens strain selection is one of the critical criteria in determining the success of transformation in plant transgenic studies. A. tumefaciens strain LBA4404 was used in this study since it exhibited a higher transformation rate in plant transgenic studies when compared to other strains, such as EHA105, C58C1, AGL1, GV3101, and GV2260 [26,27]. The transfer of T-DNA from Agrobacterium into rice calli is initiated with the attachment of Agrobacterium onto the surface of rice calli by the formation of fiber or thread-like structure, connected to each other and twinned to form a bundle of fibril material extended out from bacterium, as shown in Figure 2a. A similar structure has also been reported in different Agrobacterium-mediated plant transformations, including in carrot, wheat, and Datura innoxia [28][29][30]. The formation of fibril material allows the Agrobacterium to anchor itself onto the callus surface and promote a greater success rate of transformation. Unsystematic proliferation of the rice callus creates holes on the mucilaginous layer of the callus (Figure 2b), allowing the entrance of Agrobacterium into the inner part of the callus for T-DNA delivery.
Reporter genes are an important component in plant transgenic research, especially when assessing the integration of the gene of interest into the plant's genome at the initial stage. The GUS gene encodes the β-glucuronidase enzyme and has been extensively used in transgenic plant studies, given that the β-glucuronidase assay is sensitive and able to generate qualitative data. Histochemical data in this study showed that more than 90% of the transformed calli were evidently positive with GUS stain, and about 36.7% of the calli exhibited a deep blue staining intensity (scoring = XXX). Previous studies reported that transformation using different vector backbones such as the pPZP200 and pCAMBIA2201 produced less than 65% of GUS-positive transgenic rice calli [31,32]. This indicates that the newly constructed pMDC140-wwin2 vector in this study provides a higher transformation success rate and is able to express GUS consistently in the transformed calli.
Expression of the wwin2, hptII, and sps genes was validated using qPCR in this study. Sps was selected as an endogenous reference gene because it is highly species-specific to rice and not detected in the fifteen other different plant species tested in a previously reported qPCR analysis [20]. wwin2 encodes a PR-4 protein with chitinase properties capable of degrading chitin, a major constituent of certain fungal cell walls, such as M. oryzae. It has been proposed that the jasmonic acid defense signaling pathway is activated after pathogen challenge, which subsequently induces the production and accumulation of PR-4 proteins for local acquired resistance [33]. Several studies have also shown that over-expression of PR genes in various crops enhanced disease resistance against fungal phytopathogens [34,35]. One of the limitations of this study is that the blast resistance data and the physiological role of the wwin2 gene in the transformed rice are not available. Therefore, the defense mechanism of rice expressing the wwin2 protein towards the blast disease requires further M. oryzae-infected field data for confirmation.

Conclusions
In this study, a pMDC140 expression vector containing the wwin2 gene was constructed. The constructed plasmid showed an impressive transformation rate, with more than 90% of the transformed Tadong rice calli consistently expressing GUS. The transformation of the transgenes was confirmed using RT-PCR and the expressions of wwin2 and hptII genes were validated in the transformed Tadong rice calli using qPCR. The results from this study are beneficial for the design of M. oryzae-infected field experiments to confirm the defense mechanism of the wwin2 gene towards blast disease in rice.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11061072/s1, Figure S1: Synthesized wheatwin2 (wwin2) gene sequence in this study, Figure S2: Sequencing and BLAST analysis to confirm the integration of the wwin2 gene into the pMDC140 plasmid, Figure S3: Total RNA isolated from (a) non-transformed callus and (b) transformed callus, Figure S4: Amplification plot and C T values of the sps, hptII, and wwin2 generated using StepOnePlus TM Software v2.3.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.