Resistance gene expression in selected Indonesian pigmented rice vari‐ eties against infection by Xanthomonas oryzae pv. oryzae

Rice ( Oryza sativa L.) production is limited by bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae ( Xoo ). For decades, researchers have attempted to control this disease by growing plants with blight‐resistant Xa genes. Genetic resources often vary between rice varieties, and there is little information about the genetic resources of the pigmented rice varieties widely grown in Indonesia and their resistance genes against Xoo . The purpose of this study was to determine the expression of Xa genes in pigmented rice such as Inpari 24 and Cempo Merah (red‐pigmented) along with Hitam Bantul (black‐pigmented) and white rice varieties IR64 and Ciherang, and to evaluate their resistance to BLB. All varieties carried the Xa4 , Xa10 and xa13 genes but varied in the Xa1 , Xa7 and Xa21 genes. The rice varieties expressed some of these genes only after inoculation with Xoo . Disease assessment categorised the three different pigmented rice varieties as resistant (Ciherang, Cempo Merah and Hitam Bantul), while IR64 (white) and Inpari 24 (red) were moderately resistant. There was no specific pattern of Xa genes possession, quality of expression or resistance level to X. oryzae pv. oryzae . Therefore, when breeding plants, the selection of parental variety must be considered in terms of the possession and expression of Xa genes such as Xa10 as a molecular marker for resistance.


Introduction
Rice (Oryza sativa L.) is a vital crop consumed world wide, particularly in Asia. Several biotic factors, such as pathogens, reduce the crop production by decreasing the yield (Arshad et al. 2013). Rice farmers consider bacterial leaf blight (BLB) caused by the bacterium Xan thomonas oryzae pv. oryzae (Xoo) (Korinsak et al. 2021), the most destructive disease in rice. The bacterium can cause losses of up to 50% depending on multiple factors such as environmental conditions, growth stages, and rice varieties (Rasmiyana et al. 2019). The pathogen system ically spreads through the xylem tissue, causing wilting of the seedlings, yellowing, and leaf drying. These char acteristics make this disease especially difficult to control conventionally.
One of the strategies to control the disease is to use the resistant plant varieties produced by breeding two or more different germplasm lines (Ke et al. 2017; Tekete et al. 2020). This technique is widely used to promote the shar ing of beneficial genetic resources between germplasms, especially those conferring resistance against BLB disease resistance. This beneficial genetic resource sharing is ac complished through molecular breeding, such as molec ular assisted selection and genetic engineering (Chukwu et al. 2019; Mukhtar andHasnain 2017). Indonesia pos sesses over 17,000 rice germplasms, including wild and pigmented rice (Maulana et al. 2014; Mau et al. 2017, demonstrating diverse morphology, biochemical proper ties, and genetic characteristics. These germplasms pro vide genetic resources for rice breeding to create new va rieties with specific characteristics. Molecular genetic studies have revealed that approxi mately 44 rice genes confer resistance to various strains of Xoo (He et al. 2006; Sombunjitt et al. 2017; Neelam et al. 2020. Gene Xa1, Xa3,Xa4,Xa5,Xa7,Xa10,xa13 and Xa21 have been widely investigating and hypothesizing as genes that strongly involved with the resistance of rice against Xoo (Neelam et al. 2020). Most O. sativa subsp. japonica and O. sativa subsp. indica rice varieties pos sess these genes related to resistance, and approximately 14 genes are recessive (Neelam et al. 2020). However, al though a variety may possess Xa genes, that variety may not express the gene when challenged with Xoo. For ex ample, both IRBB1 and IR24 rice varieties carry gene Xa1, but that expression was detectable only in IRBB1 at five days postinoculation with strain T7174, a representative strain of Japanese Xoo race 1 (Yoshimura et al. 1998). Fur thermore, both IRBB5 and IRBB54 varieties carry Xa5 and Xa21 but demonstrate different expression levels (Gao et al. 2018). These results showed that varieties carrying resistance gene sequences present different expression lev els that correlate to the resistance level in BLB and disease development.
Studies in Indonesia have reported that pigmented rice (red or black) had better resistance than white rice inocu lated with Xoo. However, no data have demonstrated the expression of Xa genes in pigmented rice. Therefore, the aim of this study was to determine the expression level of Xa genes in pigmented rice varieties and evaluate their re sistance against BLB.

Growing pigmented rice seedlings
The five Indonesian rice varieties used in this study in cluded white rice (IR64 and Ciherang), redpigmented rice (Cempo Merah and Inpari 24), and blackpigmented rice (Hitam Bantul) ( Figure 1). In this study, Hitam Bantul was selected to represent the blackpigmented rice with the same germination and growth rate as well as white and red pigmented rice compared to another blackpigmented rice (Ketan Hitam). The seeds from the collection of Center of Excellence on Crop Industrial Biotechnology, Univer sity of Jember, Indonesia were treated with 10% sodium hypochlorite and rinsed twice with sterile distilled water before imbibition by deeping in sterile distillated water for one week. Seedlings were grown in a container contain ing rice field soil in the greenhouse for three weeks before genetic analysis and Xoo inoculation experiments. Each container was planted with 6 germinated seeds.

DNA genome isolation
Genomic DNA isolation was performed according to Sambrook and Russell (2001) with some modifications. Briefly, approximately 2 g of 4weekold rice seeds were ground into a powdered in liquid nitrogen. The sample was transferred and mixed with 800 μL of lysis buffer (1 M of TrisHCl [pH 8.0], 0.5 M of EDTA, and 10% of SDS) in 1.5 ml centrifuge tube, and the mixture was heated for 10 min at 65°C. After centrifugation for 10 min at 8,000 × g and 20°C, the supernatant (700 μL) was transferred into a new 1.5 mL tube, mixed with the equal volume of phenol chloroformisoamyl alcohol (PCI), and centrifuged for 10 min at 8,000 × g at 20°C. The upper layer (approximately 500 μL) was mixed with 0.1 volumes of 3 M of sodium acetate and 2.5 volumes of absolute ethanol. After 1 hour of incubation at 20°C, the mixture was centrifuged for 10 min at 10,000 × g. The precipitate was washed with 70% ethanol and dissolved in 70 μL of 1×TE buffer (pH 8.0).

Amplification of Xa genes DNA fragments
Genomic DNA fragments of eight resistance genes were detected by PCR using specific pair primers. PCR amplifi cation was performed using 80 nanograms of DNA sample in a 20µL total reaction volume, including the MyTaq HS Red Mix (Bioline, Nottingham, UK) mixture. The thirty five reaction cycles were predenaturation at 94°C for 3 min, denaturation at 94°C for 1 min, annealing for 1 min at a specified temperature (Table 1), elongation at 72°C for 1 min, and final elongation at 72°C for 10 min. The result was analyzed using a 1% agarose gel electrophoresis and was visualized under a UV transilluminator in the gel doc umentation CCD image system (Major Science, Saratoga, CA, USA).

Inoculum preparation and inoculation
The BLB pathogen, Xanthomonas oryzae pv. oryzae strain XooJ2 was grown at 28°C in a yeast extract dextrose medium for 24 h (Rejeki et al. 2021). The inoculum was prepared from a 24hour culture of X. oryzae and adjusted to a density of 10 8 CFU per milliliter. Pathogen inocula tion was performed by the leaf clipping method, as pre viously described . Briefly, sterile scis sors were dipped in inoculum suspension and used to cut the leaves of 3week seedlings 23 cm away from the tip. Plants were grown and maintained at 28-32°C in the greenhouse for symptom observation and disease assess ment.

Total RNA isolation
Leaves were harvested three days after pathogen inocu lation and used for RNA isolation. The 65 mg of frozen leaves were ground into a powder in a precooled mor tar with liquid nitrogen. Total RNA was extracted and purified using the RNAprep pure Kit (TIANGEN, Bei jing, China) according to the manufacturer's directions. The resulting total RNAs were confirmed by 1% agarose gel electrophoresis using 1× tris boric acid EDTA (TBE) buffer (pH 8.0). Total RNA quantification was performed using a NanoVue Plus spectrophotometer (Biochrom, Hol liston, MA, USA). Purified total RNA was then stored at 80°C until further analysis.

Reverse transcription-polymerase chain reaction (RT-PCR)
Since this study is earlier step on rice parental screening for molecular breeding, then we qualitatively analyzed the Xa genes expression through RTPCR. Briefly, the firststrand complementary DNA (cDNA) was synthesized from 100 ng of total RNA in 50µL total reaction volume using re verse transcriptase master mix (ReverTra Ace, TOYOBO, Osaka, Japan). The resulting cDNA was used as a tem plate for PCR amplification using a specific targetedgene primer (Table 1) in the 20µL volume of MyTaq HS Red Mix (Bioline, Nottingham, UK). Thirtyfive cycles for tar geted DNA amplification were set for denaturation at 98°C for 5 min, elongation at 72°C for 1 min, while the an nealing temperature was based on the specific primer used for the target gene (Table 1). PCR products were analyzed on 1% agarose gel with TBE buffer and were stained then visualized using a gel documentation CCD image system.

Bacterial leaf blight disease assessment
BLB leaf lesions were assessed at 0, 7, and 14 days af ter inoculation. Disease evaluation was performed using the standard evaluation system (SES) recommended by the International Rice Research Institute (IRRI 2013) by mea FIGURE 2 Agarose gel electrophoresis of PCR products from the genome of five rice varieties to detect X. oryzae pv. oryzae resistance-related genes using specific Xa-gene primers (listed in Table 1).
suring the length of the lesion. Plants were considered re sistant if the average lesion length was ≤3.0 cm, moder ately resistant if the average lesion length was 3.0-6.0 cm, moderately susceptible if the average lesion length was 6.0-9.0 cm, and susceptible if the average lesion length was >9.0 cm. The disease incidence was calculated as a percentage of the number of symptomatic plants per total number of observed plants (Rasmiyana et al. 2019).

The presence of Xa genes in pigmented rice
Xarelated DNA fragment analysis for two of white rice varieties (IR64 and Ciherang), two of red rice varieties (Cempo Merah and Inpari 24), and one of black rice va rieties (Hitam Bantul) showed that only Inpari 24 carried Xarelated DNA fragments for Xa1, Xa4, Xa7, Xa10, and xa13. All varieties contained specific DNA fragments for Xa4, Xa10, and xa13. However, DNA fragments for Xa7 and Xa21 were produced only by Inpari 24 and IR64, re spectively ( Figure 2).

The expression of Xa genes
RTPCR analysis of six Xa resistance genes in five rice varieties showed that specific Xa genes were detected in Xooinoculated plants but not in nonXooinoculated. Genes detected in rice varieties after inoculation with the pathogen included Xa7, Xa10, xa13, and Xa21. Notably, only Inpari 24 exhibited the band representing Xa1 after in oculation with the pathogen. In addition, Xa4 expression was lower in inoculated plants and was absent in all Cempo Merah both of inoculated and noninoculated plants (Fig  ure 3).

Disease assessment of pigmented rice
Although all varieties showed BLB symptoms, the inci dence varied. The highest incidence was 61.4% on IR64 and the lowest in another white rice (Ciherang), 22.7%. Blight incidence on red and black rice was moderate, 42.5-50%. Analysis of disease severity, determined by mea suring the lesion length from the leaf tip (Figure 4a), was highest for IR64 and the lowest for Hitam Bantul ( Figure  4b). Consistently, the lesion length significantly increased between 7 and 14 days postinoculation on IR64 by ap proximately 59%.

Discussion
Resistance genes (R genes) play an important role in in hibiting disease development in rice and resistance to X. oryzae pv. oryzae is associated with over 44 Xa genes (Neelam et al. 2020). The presence of these genes become an important source of genetic information when produc ing a new variety of rice, either through molecular plant breeding methods including markerassisted selection or genetic engineering (Das et al. 2017). However, the pres ence and expression of these resistance genes highly de pend on the rice varieties (Fatimah et al. 2014).
Most of the rice varieties (white, pigmentedred, and pigmentedblack) possessed multiple genes for resistance to Xoo (Figure 2). However, possessing these genes does not confer specific resistance against BLB as well as the data of lesion length and disease incidence (Figure 4). Pathotypes, environmental conditions, and gene expres FIGURE 3 Agarose gel electrophoresis of RT-PCR products from the total RNA of five (white, red-pigmented, and black-pigmented) rice varieties to detect the qualitative expression of X. oryzae pv. oryzae resistance-related genes three days after Xoo inoculation using specific Xa genes primers (listed in Table 1). White rice varieties used in this study were IR64 (lane 1) and Ciherang (lane 2); red rice, Cempo Merah (lane 3) and Inpari 24 (lane 4); black rice, Hitam Bantul (lane 5). The non-inoculated leaf treatment was sterile water. sion levels are the key factors of plant disease develop ment. Rasmiyana et al. (2019) reported that despite the presence of several resistance genes, temperature plays the most influential role in disease incidence, and subse quently, disease severity. Moreover, Sahu et al. (2020) reported that rice variety IR24 showed different genes and pathways regulation at lower temperature of 21 to 29°C compared to higher temperature of 31 to 35°C. Notwith standing, the expression level of resistance genes in rice such as Xa1, Xa7, Xa10, and Xa21 has a role in inhibiting the development of BLB in rice varieties (Yoshimura et al. 1998; Ronald et al. 2008; Wang et al. 2017. Although Ciherang and the two red rice varieties, Cempo Merah and Inpari 24, possessed Xa1, only Inpari 24 expressed the gene, but with a greater mean lesion length at 14 days after pathogen inoculation (Figure 3). The difference of expression level for each cultivar was highly influenced by the ineffectiveness of resistance gene expression to inhibit the development of Xoo that may be due to pathogen race differences. The Xa1 gene confers re sistance specifically against race 1 strains of Xoo originat ing from Japan (Yoshimura et al. 1998), while the XooJ2 strain is a species isolated from Indonesia (Rasmiyana et al. 2019). Similar to the expression of Xa1, a resis tance gene associated with transmembrane protein synthe sis (Yoshimura et al. 1998), Xa21 is also responsible for a transmembrane protein specific for Xoo isolates from race 6 strains originating from the Philippines (Ronald et al. 2008). Although the expression in IR64 increased after Xoo inoculation, it did not significantly inhibit the mean le sion length progression (Figure 3). Yoshimura et al. (1998) reported that Xa21 could be ineffective against Xoo if pro tein fail to recognize specific pathogen ligands for defense response activation.
Although IR64 and Inpari 24 had almost the same gene expression pattern, they differed in average lesion length (Figure 4). This result is likely due to Xa7, which is only present and expressed in Inpari 24 ( Figure 3). Xa7 plays a role in encoding orphans that trigger programmed cell death in rice and lead to the enduring resistance of rice to X. oryzae pv. oryzae (Wang et al. 2021). This gene is prevalent in O. sativa subsp. indica and inhibits the ex ploitation of the sucrose transporter protein (sugars will eventually be exported by Transporter14 [SWEET14]) by the pathogen through effector AvrXa7. The effector in duces the synthesis of the SWEET14 protein required for the rapid multiplication of Xoo in plants. The inhibition of SWEET14 production by the Xa7 gene product causes the infected rice to be more resistant to the pathogen (Luo et al. 2021).
Xa4 expression in inoculated rice decreased in qual ity, indicating loss of expression ( Figure 3). This result indicates that weakening the cell wall was occurring after pathogen inoculation. The Xa4 is a gene associated with cell wall synthesis, so its expression will strengthen cell walls (Hu et al. 2017). González et al. (2012) reported that Xoo breaks the integrity of the cell wall of rice by pro ducing enzymes via the type two secretion system (T2SS) resulting in the disease symptom on leaves. Decreasing the quality of Xa4 expression may cause disease incidence in all varieties (Table 2). In Ciherang and Hitam Bantul, both had similar gene expression but the lesion length (Fig  ure 4) and disease incidence (Table 2) results were slightly different from Cempo Merah. This is probably caused by the simultaneous expression of Xa4 and xa13 genes both before and after inoculation. Chu et al. (2006a) ob served that the expression of the recessive xa13 gene with Xa4 results in less resistance than in rice plants that have these genes separately. Interestingly, only these three vari eties (Ciherang, Cempo Merah, and Hitam Bantul) started to express Xa10 before pathogen inoculation (Figure 4). Xa10 is the gene that encodes the transcription activator like (TAL) effectordependent responsible for pathogen ef fector recognition . Recognition of the pathogen effector triggers the plant resistance mechanism (Zeng et al. 2015). This may affect that these varieties were less lesion than in IR64 and Inpari 24 that expressed Xa10 only after pathogen inoculation. This study suggests that the possession and expres sion of Xa genes play an important role in rice resistance. However, the expression of Xa genes, particularly Xa10 is suggested as the most considered resistance genes from among five varieties for further selection for molecular rice breeding.

Conclusions
Our results showed that the selection of source resistance is not pigmentbased but it depends on the composition and expression of the resistance genes. The results also showed that possessing resistance genes does not guaran tee the resistance of a rice variety to Xoo. Notably, pos sessing several Xa genes, particularly recessive genes, can trigger the susceptibility of rice to Xoo. Therefore, the ac curate selection of gene source and type of Xa gene is es sential for producing new rice varieties resistant to Xoo.