Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia

Abstract

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

1. Introduction

Crop diseases are one factor that can cause severe damage to agricultural production. Therefore, crop protection strategies have been developed, including breeding disease-resistant varieties and using agrochemicals to minimize damage from crop diseases. Due to agrochemicals’ high cost and environmental impact, disease-resistant varieties are more desirable. The conventional method of generating disease-resistant varieties involves introducing resistance genes to the causative pathogen from resistant plants by cross-breeding. However, this method is limited to the pathogen species for which resistance genes have been identified.

Among the pathogens with a wide host range, the necrotrophic fungus Rhizoctonia solani causes serious diseases in hundreds of plant species. R. solani is a highly destructive pathogen that causes severe symptoms in the leaves, stems, and roots of diverse plant species, including rice [1], tomato [2,3], and torenia [4,5], leading to significant economic losses. To date, no cultivar has shown strong resistance to R. solani. Additionally, there are few reports of disease-resistant genes for R. solani, and it is difficult to breed varieties resistant to the fungus in many crops.

Sugarcane smut caused by the biotrophic fungus Sporisorium scitamineum is an important disease affecting sugarcane worldwide, leading to serious losses in productivity and profitability. However, there are no effective fungicides for sugarcane smut. Sugarcane is an economically important crop for sugar production. This disease degrades the yield and quality of the raw material for sugar production. Despite the worldwide loss of sugarcane yield and sucrose caused by S. scitamineum, limited information is available regarding its pathogenic mechanisms [6]. The disease is recognized by whip-like structures produced on the terminal meristem and side shoots of infected stalks [7]. However, few effective resistant genetic resources have been reported for this fungus. Furthermore, cultivated sugarcane is an interspecific hybrid polyploid with singularly complex genomes [8], making it difficult to efficiently breed resistant varieties against the disease. Additionally, cross-breeding is limited to specific regions because it flowers only in low-latitude regions. Therefore, it is difficult to breed sugarcane varieties resistant to smut through conventional breeding methods. Based on the above reasons, genetic engineering would be advantageous in breeding smut-resistant sugarcane. However, few genes that confer resistance to S. scitamineum have been identified in plants.

Previously, we identified BROAD-SPECTRUM RESISTANCE 1 (BSR1/OsRLCK278) and 2 (BSR2/CYP78A15) by screening for resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000) and the fungal pathogen Colletotrichum higginsianum using approximately 21,000 of the rice-FOX Arabidopsis lines [9,10]. Hence, BSR1-overexpressing (OX) Arabidopsis displays resistance to PstDC3000 and C. higginsianum. Additionally, BSR1 reportedly confers broad-spectrum resistance against the bacterial [Xanthomonas oryzae pv. oryzae (Xoo) and Burkholderia glumae] and fungal (Pyricularia oryzae and Cochliobolus miyabeanus) pathogens in rice [9,11]. Therefore, the introduction of BSR1 using transgenic technology is expected to confer disease resistance to other gramineous and dicot crops.

The main objective of this study was to validate whether BSR1 overexpression could confer disease resistance in tomatoes and the ornamental plant torenia, representing dicot crops, and sugarcane, representing monocot crops. BSR1 was introduced into torenia because genetically modified (GM) ornamental crops, such as blue roses, are easily sold and more readily accepted by the general public than edible GM crops. First, BSR1 was overexpressed in tomato, torenia, and sugarcane. Next, disease resistance tests against pathogens were conducted, gross morphology was observed in each crop, and some morphological traits were examined for practical use. As a result, BSR1 overexpression successfully conferred disease resistance in three crops without detectable growth defects.

2. Results

2.1. BSR1 Overexpression Conferred Bacterial Pst DC3000 Resistance to Dicot Tomato

BSR1 overexpression increases resistance to Pst DC3000 in Arabidopsis [9]. Since Pst DC3000 was originally a tomato pathogen, tomatoes overexpressing BSR1 are expected to show resistance to the pathogen. Furthermore, the full-length cDNA of BSR1 was previously inserted into the binary vector pBIG2113SF to generate BSR1-OX Arabidopsis [9]. This construct was used to transform the tomato cultivar ‘Micro-Tom’ by the Rhizobium-mediated method. Moreover, BSR1 overexpression was confirmed in the independent transgenic tomato lines #12, #18, and #30 by quantitative real-time (qRT)-PCR (Figure S1a). The progenies were used for subsequent disease resistance assays.

BSR1-OX and wild-type (WT) tomato plants were dip-inoculated with Pst DC3000. Subsequently, three days after inoculation, the bacterial number in leaf disks (1 cm²) collected from the inoculated plants was calculated to quantify the resistance. The average bacterial numbers in BSR1-OX tomato #12 and #30 lines were significantly reduced to approximately 1/6 and 1/9, respectively, compared with WT (Figure 1). Therefore, BSR1 overexpression conferred resistance to Pst DC3000 in tomatoes.

2.2. BSR1 Overexpression Conferred Fungal R. solani Resistance to Arabidopsis and Tomato

Next, we investigated whether BSR1-OX tomatoes were resistant to pathogenic fungi. We previously reported that BSR1-OX Arabidopsis thaliana does not tolerate R. solani infection in leaves [10]. However, in tomatoes, R. solani causes damping-off disease—a root disease. Therefore, in the present study, we first examined whether BSR1-OX Arabidopsis is resistant to R. solani root infection as a pilot experiment. Two BSR1-overexpressing Arabidopsis lines (#11 and #18) were subjected to root infection of R. solani isolate (MAFF243956; AG-1 IA). BSR1-OX Arabidopsis displayed a remarkable resistance to the root infection of R. solani (Figure 2).

This result prompted us to proceed with the root infection experiment using tomatoes. For tomatoes, R. solani isolate (MAFF235116, AG-4 IIIA), whose pathogenicity is stronger than that of R. solani isolate (MAFF243956) [12], was used. BSR1-OX and WT tomato plants were inoculated with R. solani isolate using soil inoculation assay methods, as previously described [12]. As a result, more than half of the WT plants withered; however, more than half of the BSR1-OX #18 and #30 plants survived 6 days after inoculation (Figure 3a,b). The survival rates of BSR1-OX #18 and #30 (58% and 100%, respectively) were higher than those of the WT (42–44%) (Figure 3c,d). These data indicate that BSR1 can confer resistance to R. solani in tomatoes and Arabidopsis. Hence, BSR1 is expected to confer resistance to other dicot crops.

2.3. Resistance to R. solani in BSR1-OX Torenia

The soil-borne fungus R. solani causes devastating diseases in hundreds of plant species, including ornamental plants. Since BSR1 overexpression confers resistance against R. solani in Arabidopsis and tomatoes, we investigated this effect in ornamental crops. We used torenia as a representative ornamental crop because it has a simple flower structure and is easy to culture and transform [13]. The BSR1 expression vector, used for tomato transformation, was used for transforming the Torenia fournieri Lind. ‘Crown Violet’ to generate transgenic plants. As a result, overexpression of BSR1 was confirmed in two independent T0 plants (Figure S1b).

R. solani resistance assay was performed using two BSR1-OX lines (#12 and #14). The survival ratios of BSR1-OX #12 and #14 lines (100% and 75%, respectively) were significantly higher than that of the WT line (0%) 5 days after inoculation (Figure 4). Therefore, BSR1 overexpression conferred resistance to R. solani in torenia.

2.4. BSR1 Overexpression Conferred S. scitamineum Resistance to Monocot Sugarcane

The biotrophic fungal pathogen S. scitamineum causes sugarcane smut; however, the efficient breeding of varieties resistant to S. scitamineum remains a problem. Therefore, we examined whether BSR1 confers S. scitamineum resistance in sugarcane. The cDNA of BSR1 was inserted downstream of the constitutive 35S promoter with two 35S enhancers, and the construct was used to generate transgenic sugarcane lines using particle bombardment methods. After confirming cDNA insertion in the sugarcane genome by PCR, transgenic lines (T0) expressing BSR1 protein were screened by Western blot analysis. Lines 14 (lowly expressed), 30 (moderately expressed), and 35 (highly expressed) were used for the following experiments (Figure S1c).

Furthermore, axillary buds in WT and transgenic plants were inoculated with 10⁴ spores/mL S. scitamineum via needle injection. When S. scitamineum infects susceptible sugarcane plants, the fungus causes black whip-like structures in the infected plants (Figure S2) [6,14]. Therefore, the number of plants with black whip-like structures was counted, and the disease ratio was calculated. The disease ratios of BSR1-OX #14 and #30 (78% and 33%, respectively) were lower than those of the vector control (VC; 100%) and WT (93%) 150 days after inoculation (Figure 5). Additionally, the disease ratios of BSR1-OX #30 and #35 (64% and 27%, respectively) were lower than that of the vector control (100%) 130 days after inoculation in the other experiment (Figure S3). As described above, three BSR1-OX lines were resistant to S. scitamineum. Lastly, BSR1 protein expression levels correlate roughly with disease resistance levels (Figure 5, Figures S1c and S3).

2.5. Morphological Traits and Growth of BSR1-OX Plants

To breed and use the BSR1 gene, it is important that the gene does not affect other traits moreover disease resistance. Therefore, the growth and morphogenesis of BSR1 overexpressing plants were observed.

Plant height, culm height, number of tillers, and stalk diameter of three BSR1-OX lines, WT, and VC plants were measured 180 days after transplanting because such agronomic traits affect yield in sugarcane. Additionally, the leaf stage was examined to evaluate its effect on growth. Plant and culm heights of BSR1-OX #14 and #30 lines were not significantly different from those of the WT and VC lines (Figure 6a,b and Figure S4); however, those of #35 line, with the highest BSR1 expression level, were significantly lower (Figure 6a,b and Figure S1c). Therefore, a high accumulation of the BSR1 protein would have repressed normal growth. Regarding the number of tillers and stalk diameter, the values of the three BSR1-OX lines were not significantly different from those of the WT and VC (Figure 6c,d). These data suggest that BSR1-OX #14 and #30 lines had no negative effects on yield traits. Additionally, no significant difference was detected in the leaf stage among the WT, VC, and three BSR1-OX lines (Figure 6e). These results indicate that BSR1 does not affect growth and yield traits if its expression level is adequately controlled.

In tomatoes, the gross plant morphology and agronomically important fruit size of BSR1 overexpressing typical lines were similar to those of the WT tomatoes (Figure 7). Additionally, in torenia, the gross plant morphology and agronomically important flower size of BSR1 overexpressing typical lines were similar to those of the WT torenia (Figure 8). Overall, with appropriate BSR1 expression, adverse effects on growth and morphology were not observed in the tested transgenic crops (tomato, torenia, and sugarcane).

3. Discussion

3.1. Broad-Spectrum Disease Resistance by BSR1 Overexpression in Sugarcane, Tomato, and Torenia

BSR1 overexpression in A. thaliana, a model dicot plant, confers resistance to the bacterial pathogen Pst DC3000 and the fungal pathogen C. higginnsianum [9]. Additionally, BSR1 overexpressing Arabidopsis plants showed R. solani underground resistance (roots). To validate such resistance in tomato, a representative dicot crop, BSR1 was overexpressed in tomato cv. Micro-Tom. BSR1-OX tomatoes showed resistance to Pst DC3000 and R. solani. Furthermore, overexpression of BSR1 in torenia, a representative ornamental dicot crop, also confers resistance to R. solani. When BSR1 was overexpressed in sugarcane, a monocot crop, it showed resistance to S. scitamineum, a pathogenic fungus that causes sugarcane smut, the most important disease in sugarcane. Therefore, overexpression of the rice BSR1 gene would be a powerful tool to confer broad-spectrum disease resistance in dicot and monocot crops, including ornamental plants.

Plant pathogens are often divided into biotrophs, hemibiotrophs, and necrotrophs, according to their parasitic types. S. scitamineum, Pst DC3000, and R. solani used in this study were classified as biotrophs, hemibiotrophs, and necrotrophs, respectively. BSR1 overexpression conferred resistance to these three pathogens. The disease resistance mechanism of BSR1 is expected to enhance pattern-triggered immunity (PTI), elicited in the very early stages of infection when the host cells are still alive. Together, all three pathogens used in this study may have been in the biotrophic phase at the early stages of infection, when BSR1 overexpression exerted disease resistance.

3.2. Broad-Spectrum Disease-Resistant Mechanism by BSR1 Overexpression in Sugarcane, Tomato, and Torenia

BSR1 encodes a receptor-like cytoplasmic kinase, OsRLCK278, which is classified into the same RLCK-VII subfamily as Arabidopsis BIK1 [9]. We reported that BSR1 plays a role in chitin-, peptidoglycan-, and lipopolysaccharide-triggered defense responses and promotes reactive oxygen species (ROS) production [15,16]. In addition, OsCERK1, a hub-RLK, is necessary for enhanced PTI response and blast resistance by BSR1 overexpression [17]. Hence, BSR1 plays a major regulatory role in various MAMP-induced PTI responses under OsCERK1 in rice. Furthermore, since dicot Arabidopsis has CERK1 (an orthologue of OsCERK1) and BIK1 and PBL kinases homologous to BSR1, sugarcane, tomato, and torenia would have proteins and PTI systems similar to those of rice. Therefore, we speculated that BSR1 overexpression would enhance the PTI system by hijacking the innate PTI system in each crop.

The closest homologues to BSR1 in Arabidopsis are PBL19 and PBL20, which belong to the RLCK-VII subfamily [18]. Additionally, members of the RLCK-VII-4 subgroup, including PBL19 and PBL20, are involved in chitin-triggered MPK3/6, MPK4 activation, ROS production, defense gene expression, and redundantly confer basal resistance to the bacterial pathogen [19,20]. Moreover, Li et al. [21] reported that after induction by chitin, a fraction of PBL19 protein translocates into the nucleus via the N-terminal nuclear localization sequence and induces transcriptional self-amplification primarily by WRKY8. Moreover, increased PBL19 proteins interact with EDS1 and promote antifungal immunity. Although BSR1′s role has not been elucidated compared to PBL19, a common phenomenon between BSR1 and PBL19 is involved in chitin-triggered immunity and broad disease resistance against bacterial and fungal pathogens, suggesting that overproduced BSR1 protein function is similar to PBL19 or PBL19-like proteins, therefore potentiating their effects in dicot Arabidopsis, tomato, torenia, and monocot sugarcane.

Although BSR1 has been overexpressed by the constitutive CaMV35S promoter in the whole plant in Arabidopsis, the resulting BSR1-OX Arabidopsis plants did not show resistance to R. solani in their leaves [10]. However, in the present study, BSR1-OX Arabidopsis, BSR1-OX tomato, and BSR1-OX torenia showed resistance against R. solani in the root. Furthermore, Arabidopsis RLCKs, such as PBL19 and BIK1, interact with other proteins to function properly [21,22]. Notably, PBL19 is involved in immunity against Verticillium dahliae—a soil-borne fungal pathogen [21]. Therefore, an interactant of BSR1 may be present in roots but absent in leaves, leading to root-specific R. solani resistance in BSR1-OX Arabidopsis.

There are many reports of chitin-induced immunity in the rice RLCK-VII subfamily besides BSR1. Furthermore, the rice orthologue of PBL27 (OsRLCK185) [23,24,25,26], and the rice homologues of BIK1 (OsRLCK57, OsRLCK107, OsRLCK118, and OsRLCK176) [27,28], are involved in chitin-triggered signaling. BSR1 appears to be the only RLCK-VII subfamily gene reported to be overexpressed in multiple heterologous plants and confers resistance to a wide range of pathogens, as observed in this study. Since this phenotypic disease resistance trait is unique to BSR1, a specialized working mechanism would exist compared with other RLCK-VII proteins involved in chitin-triggered immunity.

Moreover, if the expression levels were appropriate, the growth and morphologies of BSR1-OX plants would be similar to those of WT and vector control plants in tomato, Arabidopsis, torenia, and sugarcane, as observed in this study. Considering the trade-off between immunity and growth [29], PTI may have a regulatory function, preventing unnecessary immunity activation. Negative regulators, such as the phosphatase CIPP1 and ubiquitin E3 ligases (PUB12 and 13), involved in PTI, have been found in Arabidopsis [30,31]. In rice, OsCERK1, OsRLCK57, OsRLCK107, and OsRLCK185 have been identified as ubiquitinated proteins after chitin induction [32]. Based on the above reports, any negative regulator which prevents prolonged activation of MAMP-induced immunity would be present in the immune mechanism of BSR1 overexpression. Moreover, the PBL19′s function is regulated by translocation into the nucleus [21]; therefore, the BSR1′s function, related to the trade-off between immunity and growth, may be regulated by nuclear translocation in each host plant. Nevertheless, the negative regulatory function of BSR1 should be elucidated to understand the reasons for the normal morphology and growth of BSR1-OX plants observed in this study.

3.3. Toward Applications for Generating Broad-Spectrum Disease-Resistant Crops

If disease-resistant genes enhance plants’ defense capacity, plant pathogens’ negative impact on agricultural productivity will be reduced without using chemicals, such as fungicides and bactericides. However, to date, the most commonly used disease-resistant genes for breeding in agriculture are R genes which induce effector-triggered immunity, and few genes involved in PTI have been used. BSR1 is one of the candidate genes involved in PTI that can be used to breed disease-resistant crops. Although detailed disease resistance mechanisms are required, our results indicate that BSR1 overexpression could be applied to different crops to confer broad-spectrum disease resistance.

4. Materials and Methods

4.1. Plant Materials and Culture

Solanum lycopersicum L. cv. Micro-Tom was used as the WT tomato. Micro-Tom (accession No. TOMJPF00001) was provided by the University of Tsukuba, Tsukuba Plant Innovation Research Center, through the National Bio-Resource Project of the AMED, Tsukuba, Japan. The WT and transgenic tomatoes were grown as previously described [12]. Moreover, Torenia fournieri Lind. ‘Crown Violet’ was used as a WT torenia material. The violet-flowered CrV cultivar selected from F1 hybrid seeds of ‘Crown Mix’ (Sakata Seed Co., Yokohama, Japan) was kindly provided by Dr. Ryutaro Aida (Institute of Vegetable and Floriculture Science, NARO, Tsukuba, Japan). One vigorously growing F1 hybrid plant was propagated vegetatively via herbaceous cutting by Dr. Ryutaro Aida and used as the experimental line in this study. The WT and transgenic torenia plants were cultured and maintained as previously described [13].

A Japanese commercial sugarcane cultivar, “KRFo93-1” (a Saccharum spp. hybrid), developed at the Kyushu Okinawa Agricultural Research Center (KARC), NARO, Japan, was used as the WT sugarcane. The WT and transgenic sugarcane were grown as follows. First, the plant stem internodes were cut and germinated under a 14/10 h light/dark regime and humidified conditions at 30 °C for 4 to 7 days. Next, the germinated nodes were used in the tests. Afterwards, for growth and morphological observations, the germinated nodes were transplanted into the soil (Bonsol No. 2, Sumitomo Chemical Co., Ltd., Osaka, Japan) and grown in a greenhouse under a natural day length for 180 days.

4.2. Plasmid Construction and Transformation

To generate transgenic tomato and torenia plants that overexpressed BSR1, the full-length cDNA fragment of BSR1 was inserted downstream of the CaMV 35S promoter at the SfiI sites of the recombinant binary vector pBIG2113SF [9] and the resulting plasmid pBIG2113SF-BSR1 was introduced into Rhizobium radiobacter strain EHA105. This construct was employed to generate lines of transgenic tomato and torenia plants using the Rhizobium-mediated method [13,33,34]. In torenia, T0 and WT plants were propagated through cuttings and used for the subsequent experiments.

For sugarcane, the HindIII-EcoRI fragment cut from pBIG2113SF-BSR1 was introduced into the multi-cloning site of the pBC-SK plasmid (Stratagene, San Diego, CA, USA), and the resulting plasmid pBC-BSR1 was used for the transformation. Additionally, transgenic sugarcane plants were produced using a PDS-1000/He helium-driven biolistic device (Bio-Rad, Hercules, CA, USA) according to the method described by Nagai et al. [35]. The basic parameters of the bombardment were the same as for the Italian ryegrass transformation [36]. All culture media used were based on the Murashige and Skoog (MS) medium [37] containing 3% (w v⁻¹) sucrose, adjusted to pH 5.8, and solidified with 0.25% (w v⁻¹) Gelrite (Wako, Osaka, Japan). For callus induction, apical meristems of glasshouse-grown plants were aseptically isolated and cultured on a callus induction medium containing 0.25 mg L⁻¹ benzyladenine, 4 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, and N6 vitamins [38] in the dark at 25 °C. Induced calli were subcultured monthly. Subsequently, the calli, approximately 3–4 months old, were divided into small pieces and arranged in a circle (3 cm in diameter) on a high-osmotic pretreatment medium containing 0.25 M sorbitol and 0.25 M mannitol and incubated for 24 h in the dark at 25 °C. Next, the calli were co-bombarded with the plasmids pAcH1 [39] and pBC-BSR1. Notably, pAcH1 and pBC-BSR1 were used to express hygromycin phosphotransferase and BSR1, respectively. Afterwards, the bombarded calli were cultured in the dark at 25 °C. After two days, the calli were transferred to a selection medium supplemented with 150 mg L⁻¹ hygromycin and subcultured to a fresh selection medium every two weeks. Moreover, hygromycin-resistant calli grown on the selection medium were cultured on a phytohormone-free regeneration medium containing 3 g L⁻¹ activated charcoal under continuous fluorescent light (40 µmol m⁻² s⁻¹) at 25 °C until green-rooted shoots were regenerated from the resistant calli. Next, the rooted plants were established in soil pods and grown in a glasshouse at 28 °C. Subsequently, the transformation was evaluated using PCR analysis with Ampdirect^® Plus (Shimazu, Kyoto, Japan) according to the manufacturer’s instructions in a ProFlex™ PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The primer sets, 5′-CGCATAACAGCGGTCATTGACTGGAGC-3′ and 5′-GCTGGGGCGTCGGTTTCCACTATCGG-3′were used to detect the 375-bp fragment of pAcH1, and 5′-AGGTGAGGTTGCACTCTGCT-3′ and 5′-ACATAGATGACACCGCGCGCGATAATTTATC-3′ to detect the 506-bp fragment of pBC-BSR1.

4.3. RNA Extraction and Quantitative Real-Time -PCR Analysis

Total RNA was extracted and purified from tomato and torenia leaves using Sepasol-RNA Super G reagent (Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s protocol. Next, first-strand cDNAs were synthesized, and qRT-PCR analysis was performed as previously described [12]. Afterwards, the BSR1 transcript levels were normalized to those of the endogenous tomato and torenia reference genes. The primers used for qRT-PCR are listed in Table S1.

4.4. Protein Extraction and Western Blot Analysis

Plant protein was extracted from sugarcane leaves and immuno-detected according to a previously described method [40]. Next, custom-made rabbit polyclonal anti-BSR1 (Biogate, Gifu, Japan) was used to detect the BSR1 protein, as described by Sugano et al. [41].

4.5. Pathogens and Pathogen Cultures

The bacteria Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was cultured using a previously described protocol [9]. The fungus R. solani was cultured on PDA agar plates (Nissui, Tokyo, Japan) at 28 °C under dark conditions for 3 days and used for disease resistance tests. The spores of the fungus S. scitamineum were provided by Shin Irei (Okinawa Prefectural Agricultural Research Center, Japan).

4.6. Bacterial and Fungal Pathogen Resistance Assay in Arabidopsis, Tomato, Torenia, and Sugarcane

The bacterial pathogen assay for Pst DC3000 in tomatoes was performed as previously described [12]. Additionally, the soil inoculation assay of R. solani in Arabidopsis, tomato, and torenia was performed as previously described [12,13].

The inoculation assay of S. scitamineum in sugarcane was performed as follows. First, the germinated nodes were used for the tests. The plant stem internodes of WT, vector control, and three BSR1-OX sugarcane lines were cut and germinated under a 14/10 h light/dark regime and humidified conditions at 30 °C for 4 to 7 days. Next, the germinated axillary buds in the nodes were inoculated with 10⁴ spores/mL S. scitamineum by needle injection. Afterwards, the internodes with-infected axillary buds were transplanted into the soil (Bonsol No, Sumitomo Chemical Co., Ltd., Osaka, Japan) and grown in a greenhouse under natural day length. Lastly, the number of plants with the disease symptom, a black whip-like structure (Figure S2), was counted 130 or 150 days after inoculation, and the disease ratio (plant number with the symptom/inoculated plant number) was calculated.