Open access peer-reviewed chapter

The Application of Genomic Approaches in Studying a Bacterial Blight-Resistant Mutant in Rice

Written By

Chang-Sheng Wang and Da-Gin Lin

Submitted: April 25th, 2016 Reviewed: December 22nd, 2016 Published: March 15th, 2017

DOI: 10.5772/67331

Chapter metrics overview

4,104 Chapter Downloads

View Full Metrics

Abstract

Rice bacterial blight disease (BBD), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the serious diseases in most rice production regions. In this report, we screened for resistance mutants from the mutation pool of TNG67 variety derived by sodium azide (SA) mutagenesis with phenotype investigation and assisted with fluorescent detection. SA0423 is a mutant of broad range resistance against Xoo for many years; the resistance was studied following the concept of central dogma. The inheritance of resistance was characterized, and three QTLs were mapped onto the genome of SA0423 using simple sequence repeat (SSR) markers and R/qtl by genomic approach. In transcriptomic approach, only one differential expression QTLs (eQTLs) were identified; two differentially expressed proteins (pQTLs) were identified and genetically characterized by proteomics after Xoo challenged in SA0423 mutant. To improve the bacterial blight resistance, makers are developed from QTLs, eQTLs and pQTLs to pyramid the resistance genes through marker-assisted breeding in our rice breeding programs.

Keywords

  • rice
  • bacterial blight disease (BBD)
  • resistance
  • mutant
  • genetics
  • genomics
  • transcriptomics
  • proteomics
  • marker-assisted breeding (MAB)

1. Introduction

Rice is a staple food crop and provides more than one-fifth of the calories to humans [1]. However, rice production is often challenged by bacterial blight disease (BBD), which is one of the most destructive diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). This disease was first found in rice by Japanese farmers in 1884. It was not a serious problem in rice production until the release of high-yielding varieties during the 1960s–1970s [24]. Some field observations displayed that this disease can lead up to 50% losses in rice planting areas [3, 5]. In Taiwan, BBD often occurs in the second crop season, and its annual pathogenesis area is usually more than 20,000 hectares, accounting for about 4% of the total rice production area. Because of climate change, this disease has become more and more serious recently [6, 7]. Furthermore, International Rice Research Institute (IRRI) proposed that BBD can cause up to 70% of yield loss when susceptible varieties are grown in the environments suitable for Xoo pathogens (http://www.knowledgebank.irri.org/index.php?option=com_zoo&task=item& item id=806&Itemid=606).

The existing prevention of BBD includes field management, fertilizer control, pesticide application and resistance varieties with the major resistance gene (R gene) or the pattern recognition receptor gene (PRR gene). In field management, appropriate spacing could prevent rice plants from the infection of pathogens. Appropriate nitrogen fertilizer application could prevent rice plants from pathogens’ infection [8]. The spray of probenazole or other chemicals might prevent the infection before transplantation, but this treatment could not be applied in tropical regions [810]. So far, the use of resistant varieties is considered to be the most effective strategy against this disease. In recent years, there is no specific bactericide which could effectively control BBD, and chemicals application also increases production cost, plant injury and environmental pollution. On the other hand, the evolution of pathogens increases the diversity and the difficulty in the breeding program for durable or broad-spectrum resistance [1113]. Therefore, breeding the bacterial blight-resistant varieties is urgently required to meet the demand of a safe rice production.

Previous studies demonstrated that climate change has been proposed to affect the microflora of Xoo in the field and even change the life cycle and evolution of Xoo pathogen. Large-scale and long-term cultivation of Xa4-mediated resistant varieties also altered the Xoo population. Consequently, resistant varieties carried with only Xa4 have become susceptible to Xoo in Southeast and South Asia [14]. Bacterial blight is one of the serious diseases often occurring in the second crop season (August to November) in Taiwan. Our previous results also displayed that the top 20 cultivars with large-scale cultivation in Taiwan were susceptible to Xoo (Table 1). Therefore, if the bacterial blight disease is endemic, it will cause serious loss to the rice production. These results indicated again that breeding of the bacterial blight-resistant varieties is urgently required to meet the demand of the Taiwanese rice industry.

Planting area during 2010–2015VarietyResponse for Xoo
OrderHaXM42XF89-b
1484,063Tai Nan No. 1177
2142,132Taikeng No. 877
3119,641Taikeng No. 1479
498,404Taikeng No. 1679
547,139Taikeng No. 999
646,204Taichung-Hsien No. 1099
741,424Taikeng No. 279
839,374Kaohsiung 13999
939,149Taikeng No. 1197
1023,767Taichung-Hsien No. 179
1122,965Taichung 19297
1219,357Taikeng No. 477
1319,108Tail Nung No. 7157
1413,989Tail Nung No. 6799
1513,867Taikeng No. 579
169341Tai Tung No. 3097
179178Kaohsiung 14577
187318Taoyuan No. 157
197152Taikeng-No. 177
205660Taichung-Hsien No. 1799

Table 1.

The resistance investigation of the top 20 rice cultivars grown in Taiwan.

Note: The resistance of the top 20 rice cultivars was investigated according to the Kauffman’s method [66]. The lesion level can be classified by a scale of five scores, such as 1 (HR), 3 (MR), 5, 7 (MS) and 9 (HS).


The availability of resistant sources is the major limitation in breeding. A series of near isogenic lines (NILs) harboured various Xa genes (IRBB NILs) that were developed on the susceptible cultivar, IR24, at the International Rice Research Institute (IRRI) [15]. The IRBB lines, often applied in the domestic resistance breeding, were introduced and inoculated with Taiwan local pathogens to test their responses in our previous work. The results indicated that only the IRBB lines carried Xa5 or Xa7 showed moderate resistance while all other IRBB lines carried single Xa gene showed susceptibility to the local pathogens (Figure 1) [16]. Many of the resistance genes were introduced into the susceptible varieties by marker-assisted selection (MAS) to improve bacterial blight resistance [17, 18]. However, many of these genes lose their resistance due to the fast evolution of pathogen [19]. It has been reported that durable or broad-spectrum resistance can prolong the bacterial blight resistance in rice [20, 21]. Actually, broad-spectrum and durable resistance can be accomplished by the introduction of one very resistance gene and pyramiding with two to three other resistance genes [22]. However, large-scale and long-term cultivation of resistant varieties might result in changes of pathogen race in the Xoo population and cause the breakdown of resistance [14, 23]. These findings indicate that exploration of new germplasms with novel resistance genes become a crucial subject in breeding resistance variety.

Figure 1.

The resistance investigation of IRBB lines against the local pathogens in Taiwan [16].

Advertisement

2. Mutant screening

Sodium azide (NaN3, SA) induced mutants can be applied to any rice breeding program at any facility, while genetically modified mutants can only be handled in the isolating facilities under the governmental regulation. A TNG67 mutant pool was developed by SA mutagenesis at the Taiwan Agricultural Research Institute (TARI) in our previous breeding program. All the mutants were screened and purified according to their morphological traits by at least 10 generations of self-crossing, selection and purification following pedigree procedures. Over 3000 pure line mutants on the same genetic background of TNG67 variety were maintained in the pool [24]. The genetic diversity of mutant lines in this pool includes disease resistance (blast, bacterial blight and sheath blight) [25], pest resistance (brown planthopper, white backed planthopper and leafroller) [26], herbicide resistance (bentazon, glufosinate and glyphosate) [27] and many agronomic traits; grain quality and morphology diversities seldom found in rice cultivars. These results suggested that the TNG67 mutant pool should have high potential in basic research as well as variety improvement [24].

To improve the bacterial blight resistance for local rice varieties, we attempted to obtain the local resistant germplasms from the selection of TNG67 mutant pool [7]. So far, at least 50 bacterial blight-resistant mutants have been selected from the mutant pool (Figure 2). These mutants might carry various genotypes of resistance and participate in the resistant pathway. Among them, SA0423 and SA0424 showed stable resistances against various Xoo pathogens for many years. The genetic analysis displayed that these two mutants might carry multiple resistant genes to confer broad-spectrum resistance and show different resistant phenotypes (data shown in the following section).

Figure 2.

The screening of resistance mutants from TNG67 mutation pool by inoculation of local pathogens in Taiwan.

Advertisement

3. Genetic and mapping of resistant genes

At present, planting resistant varieties is accepted as the most efficient, reliable and economic strategy against bacterial blight. It has been proposed that the durable and broad spectrum resistance of plants was usually governed by multiple genes or quantitative trait loci (QTLs) [28]. Therefore, the discovery of novel resistance genes against Xoo is very important in the breeding program for disease resistance. So far, 42 resistance loci (Xa) for BBD have been identified and characterized [refer to: http://www.nig.ac.jp/labs/PlantGen/english/oryzabase-e/; http://www.gramene.org/; http://www. ricedata.cn/and previous reviews] [2931]. Most of these genes were found to be controlled by dominance [32], but 14 of them, such as xa5, xa8, xa13, xa15, xa19, xa20, xa24, xa25, xa26b, xa28, xa31, xa32, xa33 and xa34, were found to be regulated in a recessive manner [33, 34]. These genes distribute among 9 chromosomes of rice genome, and 16 of them are clustered on chromosome 4 (Xa1, Xa2, Xa14, Xa31(t) and Xa38) and chromosome 11 (Xa3/26, Xa4, Xa10, Xa21, Xa22, Xa23, Xa30(t), xa32(t), Xa35(t), Xa36(t) and Xa40), respectively. At present, Xa1, Xa3/Xa26, xa5, Xa10, xa13, Xa21, Xa23, Xa25, Xa27 and Xa40 have been cloned and characterized to encode six types of proteins, i.e. NBS-LRR, receptor kinase like protein, ER membrane protein, Os8N3 protein, MtN3/saliva family member and WAK3, indicating the existence of multiple mechanisms of bacterial blight resistance in rice [3547].

Near isogenic lines (NILs) with various Xa genes on the background of IR24, a very susceptible cultivar, named IRBB NILs were applied as the donor parents [15]. Besides, molecular markers linked with Xa genes in IRBB NILs were developed through comparative mapping strategy for improving the BBD resistance of commercial cultivars [17, 18]. However, it has been reported that the plant resistance genes may breakdown due to the fast evolution of pathogen isolates [19]. Many studies suggested that large-scale and long-term cultivation of resistant varieties may result in changes of pathogen race in Xoo population and caused the breakdown of resistance [14, 23]. These findings indicated that exploration of new resistance genes has become an important subject for breeding resistance variety.

Among the previously selected resistant mutants, SA0423 shows a stable resistance to Taiwan local pathogens for years. Hence, their genetic properties and BBD resistance genes were characterized in our team. Except for the bacterial blight resistance, SA0423 also has thinner leaf blades, shorter plants, more erect plant type and less tiller number than its mutagenesis parent, TNG67 (Figure 3). A strong and stable Taiwanese epidemic pathogen, Xoo XF89b, has been used for genetic analysis and mapping the bacterial blight-resistance genes. Taichung Native 1 (TN1), a very susceptible indica rice cultivar, was used as the recipient parent. The cross TN1/SA0423 was made to generate F1 and F2 materials for genetic analysis and mapping of resistant genes. After pathogen infection, the lesion lengths of TN1, SA0423 and TN1/SA0423 F1 were 17.2 ± 1.1, 1.2 ± 0.7 and 3.4 ± 0.9 cm, respectively, indicating that the BBD resistance of SA0423 is partial dominance (Figure 4). The lesion lengths of the TN1/SA0423 F2 population showed a continuous distribution (Figure 5) and indicated that the disease resistance of SA0423 is controlled by multiple genes or quantitative trait loci (QTLs).

Figure 3.

Morphology of TNG67, SA0423 and their disease responses at 28 days after inoculation (DAI) with Taiwanese Xanthomonas oryzae pv. oryzae XF89b.

Figure 4.

Morphology of TN1, SA0423 and their F1 individual, and the disease response at 28 days after inoculation (DAI) against Taiwanese Xanthomonas oryzae pv. oryzae XF89b (A, upper panel). The lower panel of (B) shows the morphology of leaf lesion at 28 DAI; left panel shows the leaf lesion (cm) investigated at 28 DAI. Error bar is the standard error of mean (n = 3). Means with the same letter are not significantly different at 5% level by LSD test.

Figure 5.

Distribution of lesion length (cm) after inoculation with Taiwanese Xanthomonas oryzae pv. oryzae XF89b in an F2 population from the cross, TN1/SA0423.

A linkage map covering 12 chromosomes with an average distance of 11.2 cM was constructed and applied to map the resistance of SA0423 using 361 TN1/SA0423 F2 individuals [48]. QTL analysis was performed using the R program language platform (version 3.1.0; http://www.r-progect.org/) with an add-on package, qtl [46, 47]. Three QTLs are detected on chromosomes 11, 8 and 6 and account for 21.1, 11 and 9.6% of the observed phenotypic variance, respectively (Table 2 and Figure 6). Three QTLs are localized to 6, 7 and 14 cM intervals, respectively; they contribute to approximately 47% of the total phenotypic variation (resistance) and no epistatic effect could be detected among them [48].

QTLChr.QTL (Confidence interval) (cM)LODPhenotyping variance (%)Additive effectDominance effect
qBBR11.1 (Q1)11124 (121–127)26.6021.10–1.64–0.44
qBBR08.1 (Q2)839 (34–41)15.0411.04–1.20–0.82
qBBR06.1 (Q3)6120 (111–125)13.209.58–1.130.79

Table 2.

Putative QTLs were identified from the F2 population of TN1/SA0423.

Note: QTLs are labelled according to the principles of previous publications [67, 68].


Figure 6.

The linkage mapping of SSR/InDEL markers and SA0423 resistance QTLs in the F2 population of TN1/SA0423. The markers and genetic distances (cM) are labelled to the right and left of the chromosome, respectively. The QTLs are coloured with red, and other published genes and QTLs associated with BBR are labelled as blue dots and lines, respectively.

Advertisement

4. Transcriptomic studies

According to QTL analysis, all the three identified QTLs contribute to 47% of the resistance indicating that other resistance genes may exist in SA0423 [48]. Therefore, the transcriptomes of TNG67 and SA0423 were determined by microarray technologies to explore the bacterial-resistant genes in SA0423.

For a precise and non-destructive investigation in the infection process of bacterial blight pathogen after inoculation, a Xanthomonas fluorescent expression plasmid, pRBBZsGFP, was constructed with a strong fluorescent gene ZsGFP and the pBBR1MCS vector for simultaneous detection of bacterial blight pathogen infection and the gene expression [49]. Pathogens infection with XF89bZsGFP was conducted on the dark-treated albino seedlings of TNG67 rice variety; the multiplication and colonization of XF89bZsGFP could be detected in 0.5 hour after inoculation, and the maximum fluorescence was observed on the same leaf in 1 hour after inoculation (Figure 7). However, the fluorescence was reduced in the following time course indicating that the multiplication and colonization of XF89bZsGFP might be suppressed by the endogenous immune system of rice. At 7 DAI (days after inoculation), the stronger fluorescence was observed again on the same leaf and extended continuously to the leaf base, suggesting that the rice immune system was broken down by the XF89bZsGFP.

Figure 7.

Visualization of X. oryzae pv. oryzae and E. coli expressing GFP in the dark-treated albino TNG67 seedlings.

After the infection of Xoo XF89b, RNA samples prepared from the leaves of TNG67 and SA0423 collected at 0, 0.5, 1, 2 and 6 hours, respectively, were applied in the transcriptomic analysis with Agilent Oligo Microarray (60K, custom-made, Agilent Technologies) [50]. The results demonstrated that 2727, 3585 and 18,432 differentially displayed transcripts were identified in SA0423, TNG67 and in both, respectively. Among them, 58 genes involved in SA0423 resistance were further conducted by bioinformatics strategies [refer to: http://www.nig.ac.jp/labs/PlantGen/english/oryzabase-e/; http://www.gramene.org/; http://www.ricedata.cn/and previous reviews] [2931] as well as “plant-pathogen interaction” pathway (http://www.genome.jp/kegg), and clustered with BioLayout Express3D [51]. By confirming with real-time RT-PCR, 17 resistance gene candidates (Table 3) were selected for bioinformatics analysis, they have been proposed to be involved in plant-pathogen interaction pathway, biosynthetic pathway of plant hormones, autophagy and signal transduction prior to the induction of plant immune system [52].

Gene nameGene ontology
Ankyrin
Ankyrin repeat-rich protein
BPCellular process, biosynthetic process, protein modification process, post-embryonic development, anatomical structure morphogenesis, response to endogenous stimulus
MFBinding, protein binding, catalytic activity,
ATG1
ATG1
BPCellular process, cellular component organization, protein modification process
CCPlasma membrane
MFMolecular function
CaM_Chr.1-1
OsCam1-3-Calmodulin
BPBiological process, response to abiotic stimulus, post-embryonic development, signal transduction
MFBinding, protein binding
CaM Chr.1-2
OsCam3-Calmodulin
BPBiological process, response to abiotic stimulus, post-embryonic development, signal transduction
MFBinding, protein binding
CaM_Chr.2
EF hand family protein
BPProtein modification process, biosynthetic process
CCCytoplasm
MFBinding
CaM_Chr.5
OsCam2-Calmodulin
BPSignal transduction
CCPlasma membrane
MFSignal transducer activity, binding, protein binding
CMPG
Immediate-early fungal elicitor protein CMPG1
BPProtein modification process, biological process
CCIntracellular
MFCatalytic activity, binding
DUF26
Domain of Unknown
function 26-lc
BPProtein modification process, cellular process, metabolic process
CCPlasma membrane
MFkinase activity, protein binding, cellular process,
FMO
Flavin-containing monooxygenase family protein
BPCell death, signal transduction, metabolic process, response to biotic stimulus, cellular process, response to stress
CCEndoplasmic reticulum, membrane, cell
MFNucleotide binding, catalytic activity, binding
JOM
Jasmonate
O-methyltransferase
BPMulticellular organismal development, cellular process, metabolic process
CCCellular component
MFBinding, protein binding, transferase activity
PxMP
Peroxisomal membrane
protein
BPBiological process
CCPeroxisome, membrane
MFMolecular function
SAM
SAM dependent carboxyl methyltransferase
BPBiological process, cellular process, metabolic process
CCCellular component
MFTransferase activity
SNARE
SNARE associated Golgi
protein
CCCytosol
Ubi
Ubiquitin family protein
MFMolecular function
Xa2
OsSAUR21—Auxin-responsive SAUR gene family member
BPResponse to endogenous stimulus
MFMolecular function
Xa25
Nodulin MtN3 family protein
BPBiological process, cellular process, transport
CCPlasma membrane, membrane, cell
MFTransporter activity
xa5
Transcription initiation factor IIA gamma chain
BPBiosynthetic process, nucleobase, nucleoside, nucleotide and nucleic acid metabolic process
CCNucleoplasm

Table 3.

The resistance gene candidates identified from transcriptomic analysis in a bacterial blight-resistant mutant, SA0423.

Note: BP, biological process; CC, cellular component; MF, molecular function.


To confirm the function of the identified genes from transcriptomic analysis, the SSR markers flanking in 5 cM region of these genes were retrieved from GRAME web site, screened for the polymorphic markers between TN1 (the susceptible parent) and SA0423 (the resistant parent), and then genotyping was performed in the F2 population [53]. Simultaneously, the disease lesion of F2 individuals was investigated to represent the resistance phenotype after the inoculation of Xoo XF89b. The linkage between genotype and phenotype was conducted using R/qtl software by the single marker regression model. The results displayed that only RM6838 adjacent to Ankyrin showed a significantly high LOD (6.86) (Table 4) indicting that Ankyrin (LOC_Os08g15840) has a high potential to be involved in the resistance of SA0423. The bioinformatics analysis shows that this Ankyrin protein shares 76% similarity with the Arabidopsis RING type ligase, XBAT32, of an XB3 family. In Arabidopsis, Ankyrin has been proposed to negatively regulate 1-aminocyclopropane-1-carboxylate synthase (ACS), a key enzyme involved in the ethylene biosynthesis pathway, and then compromised immune system [54]. The real-time RT-PCR displayed that the expression level of Ankyrin in SA0423 was lower than that of TNG67, and higher expression levels of OsACS1 and OsACS3 were found in the BBD resistance mutant, SA0423 (Figure 8). These findings showed that ethylene metabolism may involve in the disease resistance of SA0423. A total of 15 mutations in the coding region resulting two mutation residues, Ser280Pro and Thr381Ala, were discovered in the Ankyrin of SA0423 through cloning and sequencing (data not shown). At the same time, the transgenic rice plants with less expression of ankyrin showed a significant resistance against Xoo XF89b isolate. Therefore, Ankyrin is considered to be one of the expression quantitative trait loci (eQTLs) involved in the bacterial blight resistance of SA0423.

GeneChromosomePosition (cM)MarkerLODz
CaM_Chr.1-1150.8RM60390.8941
CaM Chr.1-2150.9RM5721.7618
CaM_Chr.2225.3RM63780.0886
CMPG2131RM139380.5618
Xa24107.4RM174921.2685
JOM4120.3RM176040.5006
xa553RM177410.2725
CaM_Chr.55104.7RM69720.5717
FMO619.1RM195560.2034
SAM633.5RM2760.1920
DUF26773.2RM38260.1547
SNARE7116.1RM1362a0.2325
Ankyrin842.9RM68386.8579
ATG1073.7RM5471a0.0479
Ubi1099.8RM1470.2880
Xa251257.9RM281570.4657
PxMP1269.6RM5190.2940

Table 4.

Linkage analysis between the resistance gene candidates and the resistance trait of SA0423 by R/qtl.

Note: z LOD, log10 of odds.


Figure 8.

Quantitative analysis of mRNA expression of Ankyrin and OsACS homologs in TNG67 and SA0423 after inoculated with Xoo XF89b by using real-time RT-PCR.

Advertisement

5. Proteomics study

Proteomics technology provides a direct investigation of proteins which may participate in rice disease resistance. In previous studies, plasma membrane (PM) proteomic analysis of the genetically modified rice suspension cells with Xa21 demonstrated that PM-associated ATPase, phosphatase, hypersensitive-induced response protein, prohibitin, zinc finger/C2 domain protein, universal stress protein and heat shock protein might be involved in the early immune response against compatible and incompatible Xoos [55]. A proteomic analysis of Java 14 seedling revealed that 20 differentially displayed proteins were responded to bacterial inoculation and categorized into energy, metabolism and defence pathways [56]. These proteomic studies were conducted at 0, 12, 24 even 72 hours after inoculation [55, 56] whereas considering the rapidity of defence observed in other plant-pathogen interactions [57] and the short life cycle of Xoo, it is expected that Xoo might induce rice reprogramming immediately after pathogen infection.

A comparative proteomics analysis was conducted to characterize the proteomic profiling in leaves of TN1 (as a susceptible control), TNG67 and SA0423 after the infection of Xoo XF89b at 0, 6, 48 and 72 hours after pathogen inoculation (Figure 9). There were 60, 38 and 96 differentially displayed protein spots identified only in SA0423, TNG67 and TN1, respectively, by the separation of two-dimensional gel electrophoresis (2-DE). Finally, a total of 150 disease resistance-related proteins were identified from these protein spots through the ESI-Q-TOF mass spectrometry (MS) analyses. Ten resistance protein candidates (Table 5) were then determined by bioinformatics approach including annotation of metabolic pathway, comparative mapping analysis with published resistance loci [refer to: http://www.nig.ac.jp/labs/PlantGen/english/oryzabase-e/; http://www.gramene.org/; http://www.ricedata.cn/and previous reviews] [2931] as well as ‘plant-pathogen interaction’ pathway (http://www.genome.jp/kegg), and clustered with BioLayout Express3D [51]. These candidates were proposed to be involved in ascorbate, glyoxylate and glutathione, and oxidative phosphorylation metabolisms.

GeneMarkerhmzAhmzBhtznm(hmzA)m(hmzB)m(htz)R2AD|D/A|Fp
L-ascorbate peroxidase 1, cytosolic (APX1)RM7197303529946.568.287.570.050.8620.151580.182.50.08795
Putative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BIPM)RM5970162850948.298.8416.520.120.276–2.048317.416.050.00337* *
Glyceraldehyde-3-phosphate dehydrogenase, putative, expressed (G3PD)RM14336232340866.757.8337.40.020.5390.109490.20.770.46538
Aspartate aminotransferase (AST)RM14099422214786.619.4337.290.141.412–0.731850.526.340.00281**
2,3-bisphosphoglycerate-independent phosphoglycerate mutase, putative, expressed (BIPME)RM8084245218946.748.1346.730.050.695–0.709821.022.340.10172
Triosephosphate isomerase (TRI)RM24714163345947.438.237.010.030.401–0.815352.031.440.24307
30S ribosomal protein S4, chloroplastic (RP30S)RM5579a183640946.598.4797.050.060.943–0.483360.5130.05439
Fructose-bisphosphate aldolase, chloroplastic (FBPA)RM26143104635918.097.5967.310.01–0.247–0.533362.160.240.78348
Cysteine synthase (CYS1)RM520232843947.86.8357.80.02–0.4830.477170.990.910.40443

Table 5.

Linkage analysis between the resistance protein candidates and the resistance trait of SA0423 by MapDisto. The “**” was indicated “statistical significance” (p ≤ 0.05).

Figure 9.

2-DE image analysis of rice leaf proteome under Xoo XF89b infection. Total leaf proteins were extracted and separated by 2-DE then stained with sliver staining according to the previous protocol [58, 59]. An equal amount (200 μg) of the total proteins was loaded on each gel strip. The differentially expressed resistance-related proteins in TN1, TNG67 and SA0423 are marked as N, T and S, respectively.

The candidate genes identified from proteomics approach were genetically confirmed as previously described, the SSR markers flanking in 5 cM region of them were retrieved from GRAMENE web site, and screened for polymorphism TN1 (the susceptible parent) and SA0423 (the resistant parent). Genotyping analysis was performed in 94 TN1/SA0423 F2 individuals using the polymorphic markers. The lesion of these F2 individuals was investigated after the inoculation of Xanthomonas oryzae pv. oryzae XF89b as the resistance phenotype. The linkage between genotyping and resistance was analysed by MapDisto according to Lorieux’s protocol [60]. The result displayed that only RM5970 adjacent to the putative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (BIPM) and RM14099 adjacent to aspartate aminotransferase (AST) showed significant association with the SA0423 resistance (Table 5). BIPM has been proposed to have some important roles in glycolysis, stomatal movement, vegetative growth and pollen production in Arabidopsis [61], but it was usually found to be differentially expressed under abiotic or biotic stress [6264]. AST was found to be up-regulated in rice spotted leaf 5 (spl5) mutant that showed spontaneous HR-like lesions on its leaves, and a broadly enhanced resistance against rice blast and bacterial blight pathogens [65]. Based on these findings, BIPM and AST are found to have high potential to participate in the resistance mechanism of SA0423. These results provide novel insights into the molecular mechanisms of rice response to Xoo infection and discovery of new resistance genes as the basis for application in molecular breeding. Therefore, both BIPM and AST are considered to be the proteomic quantitative trait loci (pQTLs) for the bacterial blight resistance in SA0423.

Advertisement

6. Conclusion

Breeding resistance variety is the best strategy to overcome the bacterial blight disease damage in rice and is a very challengeable work. Availability of resistant genotype is the major limitation to the resistance improvement. However, plant disease resistance is a complex trait usually regulated by QTLs, epistatic effect, and influenced by the interactions among pathogen, host and environment.

In this review, a durable resistance mutant, SA0423, was firstly obtained from screening a sodium azide-induced mutation pool on the genetic background of TNG67 rice variety. The genomic approaches and technologies were conducted according to the concept and flow of Central Dogma. In the genomic study, the inheritance and gene corresponding to the BBD resistance of SA0423 was conducted. Linkage maps were constructed, and three QTLs (qBBR06.1, qBBR08.1 and qBBR11.1) for resistance were identified from SA0423. Meanwhile, the linkage markers for each QTL were developed according to the linkage map for marker-assisted breeding.

The transcriptomics and proteomics technologies were applied to identify the expressed genes and proteins corresponding to the pathogen inoculation for BBD resistance on SA0423. The differential displayed genes (or proteins) were annotated by blast with the gene database (NCBI and GRAMENE websites), and then their putative biological functions or the participating pathways were predicted by GO analysis. Besides, they were compared with the published resistance genes in Xa locus or putative rice ‘plant-pathogen interaction’ pathway to confirm the resistance genes or pathway in SA0423. The results demonstrated that 17 candidate genes (eQTLs) and 10 candidate proteins (pQTLs) might be involved in SA0423 resistance mechanism. The association between these candidates and SA0423 resistance was further evaluated by integration of genotyping and phenotyping of TN1/SA0423 F2 progeny through genetic approach. Both genomic and bioinformatics approaches were integrated to confirm the function and genetic relationship of the candidate genes with BBD resistance. The final results suggested that only one major expression QTLs (eQTLs) [53] and two protein QTLs (pQTLs) (Lin et al., 2017, paper in preparation ) are confirmed to confer the resistance of SA0423. It is worth to note that both the eQTLs and pQTLs identified in this study are not identified in the genetic mapping approach, and the products of eQTLs were not found in the protein profiling (pQTLs), and vice versa. These results showed that the genomic approach alone cannot unravel all the genes involved in the disease resistance of SA0423.

Phenomics or phenotype can provide the solid evidence for gene function. Our previous findings were tested through transgenic approach as well as marker-assisted backcrossing (MABC). The transgenic rice plants with less expression of ankyrin and BIPM showed significant resistance against Xoo XF89b isolate, supporting that these two eQTLs are involved in BBD resistance in rice. The identified resistance QTL, qBBR11.1, of SA0423 was introduced and improved the BBD resistance in a very susceptible indica variety, TCS10, through MABC approach. These results demonstrated that the QTLs identified from genomic, transcriptomic and proteomic approaches can be practically applied to improve the BBD resistance in rice breeding program.

References

  1. 1. Rayas-Duarte P, McGlynn WG, Stoecker BJ. Cereal foods: A full serving of nutrition. Food Sci. 2004; 25: 437–444.
  2. 2. Mizukami T, Wakimoto S. Epidemiology and control of bacterial leaf blight of rice. Ann Rev Phytopathol. 1969; 7: 51–72.
  3. 3. Mew TW, Alvarez AM, Leach JE, Swings J. Focus on bacterial blight of rice. Plant Dis. 1993; 77: 5–12.
  4. 4. Adhikari TB, Mew TW, Teng PS. Phenotypic diversity of Xanthomonas orzyae pv. orzyae in Nepal. Plant Dis. 1994; 78: 68–72.
  5. 5. Gnanamanickam SS, Brindha Priyadarisini V, Narayanan NN, Vasudevan P, Kavitha S. An overview of bacterial blight disease of rice and strategies for its management. Curr Sci. 1999; 77: 1435–1443.
  6. 6. Hsieh SPY. Rice bacterial blight. In Cheng CH, editors. Plant Protection Illustrations 8: Rice Protection. 1st ed. Taipei: Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan; 2003. pp. 317–338.
  7. 7. Wang CS, Wang AZ, Lin DG. The application of mutants in breeding disease resistance in rice. In Wang AZ, Wang CS, Ann PJ, Yu XZ, Wu HS, editors. Special Issue or the Symposium on Important Crop Pathogen Detection and Management. 1st ed. Taichung: Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan; 2013. 193 p.
  8. 8. Goto M. Fundamentals of Bacterial Plant Pathology. 1st ed. San Diego, CA: Academic Press; 1992. 342 p.
  9. 9. Lee KS, Rasabandith S, Angeles ER, Khush GS. Inheritance of resistance to bacterial blight in 21 cultivars of rice. Phytopathology. 2003; 93: 147–152.
  10. 10. Ou SH. A Handbook of Rice Diseases in the Tropics. 1st ed. Los Banos: International Rice Research Institute; 1973. 58 p.
  11. 11. Lin TF. Influence of bacterial blight on the yield and quality of rice and the breeding of resistant lines. Bull Taichung Distr Agric Improv Station. 1990; 29: 29–38.
  12. 12. Lin CS, Chang SJ. Studies on the resistance to Xanthomonas campestris pv. oryzae of rice in Taiwan I. The reaction of newly developed rice lines to Taiwanese isolates. Bull Taichung Distr Agric Improv Station. 1992; 321: 25–31.
  13. 13. Lin CS, Chen CW, Tzeng DS. The effect of microorganism bioagent for biological control of rice leaf blight disease and influence of rice agriculture characters. Bull Taichung Distr Agric Improv Station. 2003; 81: 65–77.
  14. 14. Mew TW, Vera Cruz CM, Medalla ES. Changes in race frequency of Xanthomonas oryza pv. oryza in response to the planting of rice cultivars in Philippines. Plant Dis. 1992; 76: 1029–1032.
  15. 15. Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennett J, Khush GS. Pyramiding of bacterial blight resistance genes in rice: Marker-assisted selection using RFLP and PCR. Theor Appl Genet. 1997; 95: 313–320.
  16. 16. Wang AZ, Wang CS. Genomic breeding developing rice variety with durable resistance to bacterial leaf blight and blast disease. J Agric Forest. 2009; 58: 11–24.
  17. 17. Kottapalli KR, Sarla N, Kikuchi S. In silico insight into two rice chromosomal regions associated with submergence tolerance and resistance to bacterial leaf blight and gall midge. Biotechnol Adv. 2006; 24: 561–589.
  18. 18. Sama VS, Rawat N, Sundaram RM, Himabindu K, Naik BS, Viraktamath BC, Bentur JS. A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated. Theor Appl Genet. 2014; 127: 113–124.
  19. 19. Ezuka A, Sakaguchi S. Host-parasite relationship in bacterial blight of rice caused by Xanthomonas oryzae. Rev Plant Prot Res. 1978; 11: 93–118.
  20. 20. Leung H. Stressed genomics - bringing relief to rice fields. Curr Opin Plant Biol. 2008; 11: 201–208.
  21. 21. Leung H, Zhu Y, Revilla-Molina IM, Fan JX, Chen H, Pangga I, Vera Cruz C, Mew TW. Using genetic diversity to achieve sustainable rice disease management. Plant Dis. 2003; 87: 1156–1169.
  22. 22. Li ZK, Sanchez A, Angeles E, Singh S, Domingo J, Huang N, Khush GS. Are the dominant and recessive plant disease resistance genes similar? A case study of rice R genes and Xanthomonas oryzae pv. oryzae races. Genetics. 2001; 159: 757–765.
  23. 23. Davierwala AP, Reddy AP, Lagu MD, Ranjekar PK, Gupta VS. Marker assisted selection of bacterial blight resistance genes in rice. Biochem Genet. 2001; 39: 261–278.
  24. 24. Wang CS, Tseng TH, Lin CY. Rice biotech research at the Taiwan Agricultural Research Institute. Asia Pacific Biotech News. 2002; 6: 950–956.
  25. 25. Wang AZ, Hu TK, Wang CS. Whole genome analysis of disease resistance and defense-response gene of rice. Crop, Environ & Bioinformatics. 2005; 2:267–281.
  26. 26. Tseng TH, Cheng CH, Chern CG, Yen SM, Jeng TL, Jwo WS, Wang CS. Response and genetic analysis of brown planthopper resistance in rice mutants of the TNG67 variety. Plant Prot Bull. 2003; 45: 211–223.
  27. 27. Tseng TY, Ou JF, Wang CY. Role of ascorbate-glutathione cycle in paraquat tolerance of rice (Oryza sativa). Weed Sci. 2013; 61: 361–373.
  28. 28. Johnson R. A critical analysis of durable resistance. Ann Rev Phytopathol. 1984; 22: 309–330.
  29. 29. Lin XH, Zhang DP, Xie YF, Gao HP, Zhang Q. Identifying and mapping a new gene for bacterial blight resistance in rice based on RFLP markers. Phytopathology. 1996; 86: 1156–1159.
  30. 30. Nino-Liu DO, Ronald PC, Bogdanove AJ. Xanthomonas oryzae pathovars: model pathogens of a model crop. Mol Plant Pathol. 2006; 7: 303–324.
  31. 31. Shanti ML, Shenoy VV, Lalitha Devi G, Mohan Kumar V, Premalatha P, Naveen Kumar G, Shashidhar HE, Zehr UB, Freeman WH. Marker-assisted breeding for resistance to bacterial leaf blight in popular cultivar and parental lines of hybrid rice. J Plant Pathol. 2010; 92: 495–501.
  32. 32. Verdier V, Vera Cruz CM, Leach JE. Controlling rice bacterial blight in Africa: needs and prospects. J Biotechnol. 2012; 159: 320–328.
  33. 33. Chen S, Liu X, Zeng L, Ouyang D, Yang J, Zhu X. Genetic analysis and molecular mapping of a novel recessive gene xa34(t) for resistance against Xanthomonas oryzae pv. oryzae. Theor Appl Genet. 2011; 122: 1331–1338.
  34. 34. Liu Q, Yuan M, Zhou Y, Li X, Xiao J, Wang S. A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice. Plant Cell Environ. 2011; 34: 1958–1969.
  35. 35. Chu Z, Fu B, Yang H, Xu C, Li Z, Sanchez A, Park YJ, Bennetzen JL, Zhang Q, Wang S. Targeting xa13, a recessive gene for bacterial blight resistance in rice. Theor Appl Genet. 2006; 112: 455–461.
  36. 36. Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu Z, Wang GL, White FF. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature. 2005; 435: 1122–1125.
  37. 37. Iyer AS, McCouch SR. The rice bacterial blight resistance gene xa5 encodes a novel form of disease resistance. Mol Plant-Microbe Interact. 2004; 17: 1348–1354.
  38. 38. Jiang GH, Xia ZH, Zhou YL, Wan J, Li DY, Chen RS, Zhai WX, Zhu LH. Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAγ1. Mol Genet Genom. 2006; 275: 354–366.
  39. 39. Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Holsten T, Gardner J, Wang B, Zhai WX, Zhu LH. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science. 1995; 270: 1804–1806.
  40. 40. Sun X, Cao Y, Yang Z, Xu C, Li X, Wang S, Zhang Q. Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein. Plant J. 2004; 37: 517–527.
  41. 41. Xiang Y, Cao Y, Xu C, Li X, Wang S. Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26. Theor Appl Genet. 2006; 113: 1347–1355.
  42. 42. Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang ZX, Kono I, Kurata N, Yano M, Iwata N, Sasaki T. Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA. 1998; 95: 1663–1668.
  43. 43. Tian D, Wang J, Zheng X, Gu K, Qiu C, Yang X, Zhou Z, Goh M, Luo Y, Murata-Hori M. The rice TAL effector-dependent resistance protein Xa10 triggers cell death and calcium depletion in the endoplasmic reticulum. Plant Cell. 2014; 26: 497–515.
  44. 44. Wang C, Fan Y, Zheng C, Qin T, Zhang X, Zhao K. High-resolution genetic mapping of rice bacterial blight resistance gene Xa23. Mol Genet Genom. 2014; 289: 745–753.
  45. 45. Kim SM, Suh JP, Noh TH, Reinke RF, Jena KK. Identification and fine-mapping of a new resistance gene, Xa40, conferring resistance to bacterial blight races in rice (Oryza sativa L.). Theor Appl Genet. 2015; 128(10): 1933–1943. doi: 10.1007/s00122-015-2557-2.
  46. 46. Broman KW, Wu H, Sen Ś, Churchill GA. R/qtl: QTL mapping in experimental crosses. Bioinformatics. 2003; 19: 889–890.
  47. 47. Broman KW, Sen S. A Guide to QTL Mapping with R/qtl. 1st ed. New York: Springer; 2009. 394 p.
  48. 48. Tseng HY, Lin DG, Hsieh HY, Tseng YJ, Tseng WB, Chen CW, Wang CS. Genetic analysis and molecular mapping of QTLs associated with resistance to bacterial blight in a rice mutant, SA0423. Euphytica. 2015; 205: 231–241.
  49. 49. Lin DG, Lin YL, Chen CW, Lai HC, Wang YW, Chou SY, Wang CS. Development of an efficient fluorescent visualization method for the detection of Xanthomonas oryzae pv. oryzae infection in rice planta. J Taiwan Agric Res. 2015; 64: 10–20.
  50. 50. Huang CY, Chou SY, Lee YL, Tseng WB, Chen CW, Wang CS, Lin DG. Transcriptomic analysis of a resistant mutant in response to Xanthomonas oryzae pv. oryzae Infection. J Taiwan Agric Res. 2015; 64: 145–158.
  51. 51. Theocharidis A, van Dongen S, Enright AJ, Freeman TC. Network visualization and analysis of gene expression data using BioLayout Express(3D). Nat Proto. 2009; 4: 1535–1550.
  52. 52. Panstruga R, Parker JE, Schulze-Lefert P. SnapShot: plant immune response pathways. Cell. 2009; 136: 978; e971–973.
  53. 53. Lin DG, Lin YL, Huang CY, Tseng HY, Tseng WB, Chen CW, Wang CS. Study on the resistance eQTLs in the rice bacterial blight resistant mutant, SA0423. J Taiwan Agric Res. 2016; 65: 54–69.
  54. 54. Lyzenga WJ, Booth JK, Stone SL. The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7. Plant J. 2012; 71: 23–34.
  55. 55. Chen F, Yuan Y, Li Q, He Z. Proteomic analysis of rice plasma membrane reveals proteins involved in early defence response to bacterial blight. Proteomics. 2007; 7: 1529–1539.
  56. 56. Mahmood T, Jan A, Kakishima M, Komatsu S. Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades. Proteomics. 2006; 6: 6053–6065.
  57. 57. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JD, Felix G, Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature. 2004; 428: 764–776.
  58. 58. Lin DG, Chou SY, Wang AZ, Wang YW, Kuo SM, Lai CC, Chen LJ, Wang CS. A proteomic study of rice cultivar TNG67 and its high aroma mutant SA0420. Plant Sci. 2014; 214: 20–28.
  59. 59. Lin DG, Wang CS. Extraction of total proteins from rice. Plant Bio-protocol. 2015; 4: e1277.
  60. 60. Lorieux M. MapDisto: fast and efficient computation of genetic linkage maps. Mol Breed. 2012; 31(2): 1231–1235.
  61. 61. Zhao Z, Assmann AM. The glycolytic enzyme, phosphoglycerate mutase, has critical roles in stomatal movement, vegetative growth, and pollen production in Arabidopsis thaliana. J Exp Bot. 2011; 62(14): 5179–5189.
  62. 62. Ashikari M, Wu J, Yano M, Sasaki T, Yoshimura A. Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the alpha-subunit of GTP-binding protein. Proc Natl Acad Sci USA. 1999; 96(18): 10284–10289.
  63. 63. Fujisawa Y, Kato T, Ohki S, Ishikawa A, Kitano H, Sasaki T, Asahi T, Iwasaki Y. Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice. Proc Natl Acad Sci USA. 1999; 96(13): 7575–7580.
  64. 64. Zuo J, Li J. Molecular genetic dissection of quantitative trait loci regulating rice grain size. Ann Rev Genet. 2014; 48: 99–118.
  65. 65. Chen X, Fu S, Zhang P, Gu Z, Liu J, Qian Q, Ma B. Proteomic analysis of a disease-resistance-enhanced lesion mimic mutant spotted leaf 5 in rice. Rice. 2013; 6: 1–10.
  66. 66. Kauffman HE, Reddy APK, Hsieh SPY, Merca SD. An improved technique for evaluation resistance of rice varieties to Xanthomonas oryzae. Plant Dis Rep. 1973; 57: 537–541.
  67. 67. McCouch SR, Cho YG, Yano M, Paul E, Blinstrub M, Morishima H, Kinoshita T. Report on QTL nomenclature. Rice Genet Newslett. 1997; 14: 11–13.
  68. 68. Tabien R, Li Z, Paterson AH, Marchetti MA, Stansel JW, Pinson SRM. Mapping QTLs for field resistance to the rice blast pathogen and evaluating their individual and combined utility in improved varieties. Theor Appl Genet. 2002; 105: 313–324.

Written By

Chang-Sheng Wang and Da-Gin Lin

Submitted: April 25th, 2016 Reviewed: December 22nd, 2016 Published: March 15th, 2017