The Role of Nanochitosan on the Expression of Rice Resistance Genes against Bacterial Leaf Blight

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) on rice, is an important disease that has been reported to reduce production globally (Adhikari et al., 1995). Yield loss due to this disease is estimated to reach 20-50% on severely infected fields and 10–20% if infection occurs at maximum vegetative stages (Wang et al., 2005). The most used management practice for Xoo is to use resistance varieties; however, the high evolution rate of this pathogen has caused this pathogen to overcome plant resistant characters (Suryadi et al., 2011; Joko et al., 2019). Combining various management technique may serve as a solution to manage the evolution issue to this pathogen. Chitosan has been recently developed as a plant elicitor, which is considered to be economically effective and able to induce rice resistances against BLB (Modina et al., 2009). Chitosan may act as a pathogen/microbe-associated molecular pattern (PAMP/MAMP) in various pathosystems. PAMP/MAMP may be effectors that is secreted by pathogens. Chitosan will be recognized by plant pattern recognition receptor (PRR) and cause resistant responses from plants (Hadrami et al., 2010). Chitosan in large amounts are difficultly dissolves in liquid solvents setting a challenge when applied on fields. A solution to this challenge, is by formulating chitosan into nanoparticles. Nanoparticlesized chitosan (CNP) has been used in agriculture due to its biodegradability, solubility, high permeability, non-toxic effects on humans, low prices, and effectivity compared to larger-sized chitosan (Manikandan & Sathiyabama, 2015). This study was conducted to determine the effect of CNP solution on the expression of the resistant genes, Xa21 and Xa1, on rice variety IR64, which is susceptible against Xoo. ABSTRACT


INTRODUCTION
Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) on rice, is an important disease that has been reported to reduce production globally (Adhikari et al., 1995). Yield loss due to this disease is estimated to reach 20-50% on severely infected fields and 10-20% if infection occurs at maximum vegetative stages (Wang et al., 2005).
The most used management practice for Xoo is to use resistance varieties; however, the high evolution rate of this pathogen has caused this pathogen to overcome plant resistant characters (Suryadi et al., 2011;Joko et al., 2019). Combining various management technique may serve as a solution to manage the evolution issue to this pathogen. Chitosan has been recently developed as a plant elicitor, which is considered to be economically effective and able to induce rice resistances against BLB (Modina et al., 2009).
Chitosan may act as a pathogen/microbe-associated molecular pattern (PAMP/MAMP) in various pathosystems. PAMP/MAMP may be effectors that is secreted by pathogens. Chitosan will be recognized by plant pattern recognition receptor (PRR) and cause resistant responses from plants (Hadrami et al., 2010). Chitosan in large amounts are difficultly dissolves in liquid solvents setting a challenge when applied on fields. A solution to this challenge, is by formulating chitosan into nanoparticles. Nanoparticlesized chitosan (CNP) has been used in agriculture due to its biodegradability, solubility, high permeability, non-toxic effects on humans, low prices, and effectivity compared to larger-sized chitosan (Manikandan & Sathiyabama, 2015). This study was conducted to determine the effect of CNP solution on the expression of the resistant genes, Xa21 and Xa1, on rice variety IR64, which is susceptible against Xoo.

Xanthomonas oryzae pv. oryzae Culture Preparation
The X. oryzae pv. oryzae strain used was obtain from the culture collection of Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta. The Xoo MAG 2 was isolated from infected rice, Ciherang variety, collected from Magelang, Central Java. Isolates were grown on solid peptone sucrosa agar (PSA) and incubated at 28 o C for 3 days in order to activate Xoo (Joko et al., 2000). Solid PSA medium (pH 7.0) per liter contained 5 g of peptone; 0.5 g of K 2 PO 4 ; 0.25 g of MgSO 4 .7H 2 O, and 20 g of sucrose.

Rice Plants and Nanoparticle-sized Chitosan Application
This study was done from February to November 2018 at Reseach Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta. Rice seeds, variety IR64 and labelled as stock seeds, were obtained from Yogyakarta Assessment Institute for Agricultural Technology (BPTP) Yogyakarta. The experiment consisted of 4 treatments: 1) mock: plants were inoculated with sterile distilled water and without CNP applications; 2) a positive control/C(+): plants were inoculated using Xoo and without CNP applications; 3) without CNP application/CNP(-): plants were inoculated with Xoo and without CNP; 4) with CNP/CNP(+): plants were inoculated with Xoo and applied with CNP. Each treatment was replicated 4 times with each pot containing 2 rice plants. As much as 30 mL of CNP were applied according to each treatment (Modina et al., 2009). CNP solvents were made using an ionic gelation method (Handani et al., 2017) at concentration of 0.065%, pH 3.31 and particle size of 150.2 nm based on the Particle Size Analyzer (PSA). This is caused due solvents agglomerated after being left for a couple of hours at concentration of < 0.06%. Therefore, solubility was not stable based on this experiment. CNP were applied weekly starting from rice seedlings 2 to 10 weeks after transplanted from seedbeds and grown for 2 weeks. The negative and positive control were applied with sterile distilled water with similar volumes and application intervals.

Xanthomonas oryzae pv. oryzae Inoculation on Rice Plants
Inoculums of Xoo were subcultured on liquid peptone sucrose medium and incubated at 28 o C for 2 days. The suspensions of Xoo turbidity or density were measured before inoculation using a spectrophotometer at OD 600 = 0.5 or equivalent to colony density of ± 242 × 10 6 cfu/ml. Inoculation of BLB causing pathogens were done using the leaf clipping method (Yinggen et al., 2017) when rice were 41 days old. Similar clipping method were done on mock and K (+) with sterile distilled water.

Analyzing Expression of Resistant Genes, Xa21 and Xa1, of Rice
Total RNA was extracted from leaves with and without CNP treatments at 0 days and 4 days after Xoo inoculation. RNA was extracted from plant tissue using Rneasy Plant Mini Kit (Qiagen). Isolated RNA was then synthesized into cDNA using cDNA kits. The resistant genes Xa21 and Xa1 were first confirmed using conventional PCR reaction to determine the existence of both genes and optimum annealing temperature for real time PCR. Annealing temperature and time for Xa21, Xa1, and ubiquitin, a gene used for internal kontrol, respectively were 61 o C for 45 s, 59 o C for 45 s, and 55 o C for 30 s (Sutrisno et al., 2018). Real Time PCR (Bio-Rad CFX96) were done according to protocols and replicated twice for each sample. Threshold cycle (Ct) values from results of targeted genes form real time PCR were normalized with ubiquitin Oryza sativa CT value and analyzed using a livak method (Livak & Schmittgen, 2001). Value 2^-ddct from the calculations show the folds of change of gene expressions. Primers used for real time PCR (Table 1) specificity were checked through https://www.ncbi.nlm.nih.gov/ tools/primer-blast/primertool.cgi.

Bacterial Leaf Blight Symptoms Observation
Observation of BLB symptoms caused by Xoo were defined in percentages of disease intensity (Strange, 2003) and Area Under the Disease Progress Curve (AUDPC) were calculated using formulas as described by Ahmed et al. (1999). Morphological symptoms from leaf blight lesions were observed according to Rusli et al. (2016). Scale of BLB infection were determined based on Standard Evaluation System (SES) (IRRI, 2013). Percentages of leaf area infected were determined as disease incidences and calculated as described by Wheeder (1969). BLB symptoms were measured at 1, 2, 3, and 4 weeks after Xoo inoculation. Observation data were analyzed using Mann-Whitney SPSS to determine significant differences at α=0.05.

RESULTS AND DISCUSSION
All cDNA samples from all treatment tested reverse transcriptase RNA were confirmed to contain the expression of Xa21 (Figure 1). Expression of Xa21 from all treatments indicated that the expressions of this gene is related to either inoculation using sterile distilled water, Xoo, or CNP application. Leaves that were not treated with Xoo still expressed Xa21; therefore, Xa21 is constitutively expressed by leaf tissue. The gene Xa21 is expressed from resistant and susceptible variety, but not determine by the infection of Xoo or mechanical damages (Century et al., 1999).
The Xa1 gene was also expressed from all cDNA reverse transcriptase RNA samples from all treatments (Figure 1). Different from Xa21, Xa1 expression is induced from Xoo inoculation or mechanical damages (Yoshimura et al., 1998). The expression of Xa1 from the mock and CNP(-) is induced due to mechanical damage from the clipping method.
Results of disease intensity showed that intensities were lower on plants treated with CNP compared to the C(+) only on week 4 (Figure 2), implying that application using CNP 8 times (4 times after inoculated with Xoo) with an 7 day interval was sufficient to decrease BLB intensity. This may be caused by the low concentration of CNP or time intervals between application were too long after inoculation with Xoo. The Xa21 gene is fully expressed at tillering (Park et al., 2011). Therefore, it would be more beneficial if CNP applications to induce resistant genes were done after rice plants reach maximum tillering. The AUDPC value from the positive control was not   The 2^-ddct value for variety IR64 showed an increase of Xa21 resistant gene after CNP application for CNP (-) for 1.18-folds at day 0 and 1.31-folds at 4 days compared to the mock. The Xa21 gene experiences increasement that were higher on day 4 for CNP (-) compared to day 0 ( Figure 3). This showed that CNP application was able to increase the expression of Xa21 on rice variety IR64, however without Xoo inoculation.
When inoculated with Xoo, the expression of Xa21 was lower based on its 2^-ddct being < 1, however 2^-ddct value of CNP(+) on day 4 was larger than day 0, whereas smaller on C (+) (Figure 3). Rice varietas IR64 is a susceptible variety to BLB (Wahab et al., 2017), which explain the decrease of Xa21 gene expression on day 4 after inoculated with Xoo, however the decrease was lower when applied with CNP. This showed that CNP was able to decrease the downregulation of Xa21 gene expression on rice variety IR64 inoculated with Xoo.
Xa21 expression from the C(+) treatment at day 4 was smaller compared to day 0 ( Figure 3) and may be caused by the low content of XA21 binding protein 3 (XB3) and high content of XB15 in plants. Protein XB3 contains an Ring Finger (RF) that interacts with the XA21 kinase domain. XB3 is specifically transphosphorylated by XA21 kinase domain. Reduction of XB3, triggers a decrease of XA21 protein and plants resistances related to XA21, therefore positively act in plant immunity mediated by XA21. Accumulation of XA21 protein complex requires XB3, however XB3 does not require XA21 for stability. If silencing of XB3 occurs, concentration of XA21 protein will decrease; however, XB3 protein accumulated in plants were similar whether Xa21 gene were present or not (Wang et al., 2006). XA21 binding protein 15 (XB15) is Protein Phosphatase 2C (PP2C), a group of serine/threonine phosphatase which acts as a monomer that requires Mn 2+ and/or Mg 2+ to regulate negative immunity. Dephosphorylate kinase by phosphatase protein is a common mechanism to decrease signaling through kinase. Over-expression of XB15 decreases XA21 mediated resistances against Xoo. XB15 is related to serin on JM (JuxtaMembrane) XA21 and synthesis of XA21/XB15 complex induced by Xoo detected 12 hours after inoculation and increase significantly after 24 hours (Park et al., 2008).
Based on the research done by Akamatsu et al. (2016), the concentration of CNP used in this study was enough to induce expressions of genes related to resistance of rice plant cells. The same research also stated that chitosan concentration > 15 μg/mL was able to induce the production of reactive oxygen spesies (ROS) and other genes related to rice resistance. Therefore, the development of BLB may be caused by other factor, such as pH. Nanoparticle-size chitosan pH solvent reached acid, specifically 3.31. This acid condition may affect week 1-4 and AUDPC on rice variety IR64; C = Control, CNP = Chitosan Nanoparticles, (+) = inoculated using Xanthomonas oryzae pv. oryzae; different letters indicated significant differences at α=5% (Mann-Whitney test) Mg 2+ uptake by rice plants. Ion uptake is affected by pH of external solvent. The level of Mg 2+ uptake may double at pH value of 4.5 compared at pH 6.5 after 15 minutes observations (Kobayashi & Tanoi, 2015). This causes acid condition to increase Mg 2+ uptake by rice plants. Over-expression of XB15 decreases resistances mediated by XA21 against Xoo (Park et al., 2008). Therefore, external solvent which are acid may supply Mg 2+ for XB15 activation.
The application of CNP that were not able to increase Xa21 expression of plants inoculated with Xoo may be due to the effector Xoo2875, effector type III of Xoo which may help suppress rice resistance. Yamaguchi et al. (2013) reported that disease lesions were not able to develop on wild-type rice inoculated with the mutant Xoo hrpX deficiency-T3SS, a mutant that disallow sending of effector type III. This mutant is able to strongly induce Pattern Triggered Immunity (PTI) on rice. On the other hand, transgenic rice that expressed Xoo2875 (Xoo2875-OX) demonstrated severe lesions. Population of mutant Xoo hrpX on plant Xoo2875-OX were also 100-folds compared to wild-types (Yamaguchi et al., 2013). Besides Xoo2875, effector type III that are able to suppress plant resistance are for example AvrPto (Xiang et al., 2008) and AvrPtoB (Wang et al., 2019) on Pseudomonas syringae. The expression of Xoo2875 suppressed Brassinosteroid (BR) and MAMP response of plant inoculated by the mutant Xoo hrpX. Xoo2875 hinders resistance against Xoo dan BR response by downgrading the function of OsBAK1. Besides interacting with OsBAK1, Xoo2875 also interact with OsBiSERK1, which is close to OsBAK1 and involved in immune responses. However, Xoo2875 does not interact with OsBRI1 or Xa21. BAK1 is a common component of many MAMP receptor complex; thus, suppression the BAK1 functions is an effective strategy of PRR due to Xoo2875 blocking MAMP signaling pathway (Yamaguchi et al., 2013). Long et al. (2018) reported that combination of 3 Xanthomonas outer protein (Xop), including XopN, XopV, and XoZ as a type III non-TALE (transcription activator-like effectors) effector of Xoo, was able to repress mitogen-activated protein kinase (MAPK) activity and has a role in virulence when Xoo infect and forms lesion symptoms. MAPK is a PTI response due to peptidoglikan, a common PAMP molecule on Xoo.
The 2^-ddct value showed an increase of Xa1 expression on plants treated with CNP and inoculated with sterile distilled water by 4.27-folds compared to the negative control at day 0 ( Figure 3).
Expression of Xa1 decreased on rice plant treated with CNP and Xoo and measurements from day 4 were slightly larger than day 0. However, 2^-ddct values from C(+) on day 4 were smaller than day 0 ( Figure 3). This implies that CNP can decrease the downregulation of Xa1 expression of rice variety IR64 after inoculated with Xoo even though only slightly. The gene Xa1 will recognize whole TALE of Xoo as an avirulen effector. Previous TALE variants are named pseudogene, also named interfering TALE (iTALE). iTALE is a mutant TALE on their stop codon premature or large deletions on the 3' end for code sequence, but expressed as a sliced protein on the C end. Xoo mutates a couple of TALE to not only avoid protein recognition of Resistance NLR, but also actively push resistance through progenitor TALE on compatible interaction (Sasaki & Ashikari, 2018).
Rice variety IR64, a susceptible variety to BLB, minorly expressed Xa1 gene after inoculated with Xoo in both CNP and non-CNP treated plants, due to Xoo ability to avoid whole TALE recognition from Xa1 through clipped iTALE/TALE; thus, suppressing activity of the resistant gene Xa1. This caused compatible interaction and CNP solvent in this study to not yet be able to suppress the effect of iTALE from Xoo.

CONCLUSION
Nanoparticle-sized chitosan were able to increase the expression of the resistant genes Xa21 and Xa1 on rice. However, expression increase was suppressed by Xoo. Studies regarding other genes that affect rice immunity against Xoo and virulent gene of Xoo are required. This will help the effectivity of CNP solvents in more detail as a base to development of chitosan nanoparticle in expressing resistant genes against BLB.

ACKNOWLEDGEMENT
Authors would like to thank KOPPERT B.V. for providing funding for this research.