2. Genetics of BB resistance
Genetic analysis of many plant-pathogen interactions has demonstrated that plants often contain a single locus that confers resistance against a complementary avirulence gene . The genetic basis of host resistance to BB has been studied in depth. The genetics of resistance to bacterial blight was first carried out by Japan and IRRI, subsequently, followed by Sri Lanka, India, China, and so on. As there is diversity of Xoostrains in different countries, scientists found that it was difficult to characterize and distinguish the resistance genes. In order to compare the identified genes, the identical differential standard was set up . About 42 BB resistance (R) genes, designated from Xa1to Xa42[5, 17, 18], conferring resistance against various strains of Xoo, have been identified from cultivated, mutant population, and wild rice species (Table 1). These genes have been mapped on six of the twelve rice chromosomes. A total of 14 recessive genes (xa5, xa8, xa13, xa15, xa19, xa20, xa24, xa25, xa26b, xa28, xa31, xa32, xa33, and xa34) in the series from Xa1to Xa42have been identified. Of these Rgenes, nine of the Rgenes have been cloned and characterized (Xa1, Xa3/Xa26, xa5, xa13, Xa10, Xa21, Xa23, xa25, and Xa27) encoding different types of proteins, suggesting multiple mechanisms of R-gene–mediated Xooresistance [7, 8, 10, 11, 13, 14, 19–21]. Most of these genes provide complete and race-specific resistance to Xooand have been used singly or in combination in rice breeding for BB resistance [22–32]. Since the bacterial races tend to evolve continually, influenced by the artificial and natural selection of genes resistant to BB, it is required to explore critically the new resistant resources to combat the evolved races.
|Xagene||Resistance to Xoorace||Donor cultivar||Chromosome||Reference|
|Xa1||Japanese race-I||Kogyoku, IRBB1||4||[10, 33, 34]|
|Xa2||Japanese race-II||IRBB2||4||[33, 36]|
|Xa3/Xa26||Chinese, Philippine, and Japanese races||Wase Aikoku 3, Minghui 63, IRBB3||11||[9, 37]|
|Xa4||Philippine race-I||TKM6, IRBB4||11||[39, 40, 45]|
|xa5||Philippine races I, II, III||IRBB5||5||[11, 40, 46]|
|Xa6||Philippine race 1||Zenith||11|||
|xa9||Philippine races||Khao Lay Nhay and Sateng||11|||
|Xa10||Philippine and Japanese races||Cas 209||11||[54, 55, 57]|
|Xa11||Japanese races IB, II, IIIA, V||IR8||3||[58, 59]|
|Xa12||Indonesian race V||Kogyoku, Java14||4|||
|xa13||Philippine race 6||BJ1, IRBB13||8||[14, 61–64]|
|Xa14||Philippine race 5||TN1||4||[65–67]|
|xa15||Japanese races||M41 mutant||–|||
|Xa18||Burmese races||IR24, Miyang23, Toyonishiki||–|||
|xa19||Japanese races||XM5 (mutant of IR24)||–|||
|xa20||Japanese races||XM6 (mutant of IR24)||–|||
|Xa21||Philippine and Japanese races||O. longistaminata, IRBB21||11||[7, 76, 77]|
|Xa22||Chinese races||Zhachanglong||11||[79, 80]|
|Xa23||Indonesian races||O. rufipogon(CBB23)||11|||
|xa24(t)||Philippine and Chinese races||DV86||2||[81, 82]|
|xa25/Xa25(t)Xa25||Chinese and Philippine races||Minghui 63, HX-3 (somaclonal mutant of Minghui 63)||12||[85, 86]|
|xa26(t)||Philippine races||Nep Bha Bong||–|||
|Xa27||Chinese strains and Philippine race 2 to 6||O. minutaIRGC 101141, IRBB27||6||[26, 85]|
|xa28(t)||Philippine race2||Lota sail||–|||
|Xa29(t)||Chinese races||O. officinalis(B5)||1|||
|Xa30(t)||Indonesian races||O. rufipogon(Y238)||11|||
|Xa32(t)||Philippine races||Oryza australiensis(introgression line C4064)||11|||
|Thai races||Pin Kaset|
|Xa35(t)||Philippine races||Oryza minuta(Acc. No.101133)||11|||
|Xa38||Indian Punjab races||O. nivaraIRGC81825||–||[17, 96]|
|Xa39||Chinese and Philippines races||FF329||11|||
|Xa40(t)||Korean BB races||IR65482-7-216-1-2||11|||
|xa41(t)||Various Xoostrains||Rice germplasm||–|||
|xa42||Japanese Xooraces||XM14, a mutant of IR24||3|||
Summary of resistance genes to bacterial blight in rice.
Revised and updated from Ref. .
The Xa1gene was identified by Sakaguchi  from rice cultivars Kogyoku and Java14. It conferred a high level of specific resistance to Japanese Xoorace 1. Since then, Xa1has been extensively used in Japanese rice breeding as Xoorace 1 is the most prevalent in Japan. Later on, a high-resolution genetic map was developed for Xa1using F2 population and RFLP markers . Three markers (XNpb235, XNpb264, and C600) on chromosome 4 were found to be tightly linked to Xa1, while another RFLP marker U08750 was at 1.5 cM distance from Xa1. Screening of the YAC library with three linked markers resulted in identification of ten contiguous YAC clones. Out of these clones, Y5212 clone hybridized with all the three markers and was confirmed to possess the Xa1locus. Sakaguchi  also identified another BB resistance gene, Xa2, in rice cultivar Tetep. It conferred specific resistance to T7147 (Japanese Xoorace 2). Earlier, Xa2was mapped on chromosome 4, linked to Xa1with a recombination frequency of 2–16% . He et al.  reported the fine mapping of Xa2using an F2 population of ZZA (Zhengzhui Ai) X IRBB2 and Xa2 was localized between two markers spanning approximately 190 kb region. Sequence analysis of this region revealed the presence of a homologous sequence of leucine-rich repeat (LRR) kinase, which is the product of a cloned BB resistance gene, Xa21 .
The Xa3gene was identified in a japonicavariety Wase Aikoku 3 . It was mapped to the long arm of chromosome 11 and was found to be tightly linked to another BB resistance gene Xa4 [38, 39]. Xa3was later known to be genetically linked to Xa26, another BB resistance gene. For characterization of Xa3, the gene was fine mapped using a population segregating for only a single resistance gene and markers developed from Xa26gene family. Genetic analysis showed that Xa3co-segregated with the Xa26gene marker but segregated from the markers of other members of Xa26gene family.
DNA fingerprinting also revealed that IRBB3 carrying Xa3had the same copy numbers of Xa26gene family members as were present in Minghui 63 carrying Xa26. The putative coding products of Xa3/Xa26and the susceptible allele xa3/xa26shared 92% homology with difference in the LRR domains, proving that Xa3is the same as Xa26.
The Xa4and xa5genes were identified by Petpisit et al. . The dominant gene Xa4confers resistance to Philippine race 1 of Xooand was identified in rice cultivars TKM6, IR20, IR22, and IR72. The recessive gene xa5confers resistance to Philippine Xooraces 1, 2, 3, and 5 . Yoshimura et al.  reported the mapping of Xa4and mapped an RFLP marker G181 on one flanking side of Xa4locus on chromosome 11. However, no marker was identified on the other flanking side of Xa4locus. The Xa4locus was further mapped between two RFLP markers, RZ536 and L457 . Wang et al.  localized the Xa4locus between G181 and L1044 at a distance of 4.4 and 3.8 cM, respectively. However, using other rice molecular linkage maps as references [43, 44], Sun et al.  deduced that Xa4locus reported earlier [41, 42] was not in the same position. Using an F2 population from IR24 X IRBB4 cross, Xa4gene was genetically mapped to a region less than 1 cM. A contig map was constructed consisting of six nonredundant BAC clones, spanning approximately 500 kb length. Analysis of recombination events located Xa4locus to one BAC “3H8” and assay of recombinants using subclones of “3H8” along with sequence analysis, further narrowed the Xa4locus to a 47 kb fragment.
A high-resolution genetic map of the chromosomal region harboring xa5was developed and mapped to a 0.5 cM interval between markers RS7 and RM611, which spanned an interval of 70 kb and contained 11 open reading frames. The potential candidate gene products of xa5may be basal transcription factor IIA (TFIIA), an ABC transporter, a tRNA synthase, a MAP kinase and a cysteine protease, as well as four unknown, hypothetical or putative proteins .
While studying the inheritance of resistance to Xooisolate PXO61 in five rice cultivars, Sidhu and Khush  observed that the same gene conferred resistance in all the cultivars. The pattern of segregation indicated a monogenic recessive factor when plants were inoculated at booting stage, but monogenic dominant when plants were inoculated during flowering. This reversal of dominance was caused by dosage effect of the resistance gene at a specific stage of growth of the plant when exposed to the bacterial inoculum. Apparently, the gene could express itself at booting stage only when present in two dosages. Heterozygous plants with one dose of resistance allele were susceptible during booting. However, at flowering, one dose was enough for expression of resistance. The new gene identified as Xa6and was linked to Xa4with a crossover value of 26%. Later studies on the genetics of resistance to Xooin 74 cultivars of O. sativausing PXO61 isolate from the Philippines identified an additional dominant gene, Xa7, that conferred resistance in DZ78 . A recombination value of 8.8% between Xa7and G1091 located at 107.5 cM on chromosome 6 was determined . Porter et al.  performed the AFLP analysis of a segregating, near-isogenic F3 population of IR24 X IRBB7, which revealed one polymorphic fragment, M1, mapped to position 107.3 cM on the rice map. Sequence comparisons of resistant and susceptible lines near M1 were used to generate additional markers linked to the rice BB resistance gene Xa7. A sequence-tagged site (STS) named M2 was mapped proximal to M1 and farther from Xa7, indicating that Xa7lay distal to M1. Two SSRs, M3 and M4, were mapped at a distance of 0.5 and 1.8 cM from Xa7, respectively. The pattern of recombinants and the map distances indicated that Xa7was located in the region corresponding to the ends of physically mapped Clemson University Genomics Institute (CUGI) BACs 96 and 143. A complex repeat was identified in the DNA sequence from O. sativacultivars 93-11 and Nipponbare that matched the end of contig 96 and an EST marker (C52865S). Amplification of the repeat revealed the presence and absence of the repeat in IR24 and IRBB7, respectively. However, no recombinants were identified between Xa7and the polymorphic repeat (M5). Comparison of the physical and genetic maps of rice in that region indicated that Xa7lay within 40 kb of M5, a distance suitable for MAS and cloning of Xa7. A high-resolution genetic map of the chromosomal region surrounding Xa7gene was constructed using SSR markers from the Gramene database (www.gramene.org/db) based on the Xa7gene initial mapping on chromosome 6 . Primary analysis of F2 population from the cross of IRBB7 and IR24, located Xa7in approximately 0.28 cM region. To walk closer to the target gene, recombinant F2 individuals were tested using STMS markers and the gene was finally mapped to a 0.21 cM region between markers GDSSR02 and RM20593. A contig map corresponding to Xa7gene was constructed using reference sequence of cultivar Nipponbare through bioinformatics analysis. The target gene was assumed to span an interval of approximately 118.5 kb, containing a total of fourteen genes. Candidate gene analysis showed that the fourteen genes encoded novel domains that had no amino acid sequence similar to other cloned Xa(xa) genes.
A single recessive gene, xa8, conditioned resistance in rice germplasm accession PI 231129. Vikal et al.  mapped the gene between two consecutive SSR markers, RM21044 and RM21045 at 7.0 and 9.9 cM, respectively, on chromosome 7 and the physical distance between markers was 9.5 kb. This region harbors three intact genes, which codes for putative expressed proteins viz., Zinc finger A20 and AN1 domain-containing protein (LOC_Os07g07400), oxidoreductase, 2OG-Fe oxygenase family protein (LOC_Os07g07410), and Gibberellin 20 oxidase 1-B protein (LOC_Os07g07420). All the three candidate genes are responsible for plant response to various stresses.
The BB resistance in ‘Khao Lay Nhay’ and ‘Sateng’ was recessive in nature and were allelic to each other but nonallelic to and segregated independently of Xa3, Xa4, xa5, Xa7, and xa8. This new recessive gene was linked to Xa6with a crossover value of 5.9%. This new gene for resistance was designated as xa9. The Xa10was identified from rice cultivar Cas 209 [54, 55]. There is an extensive polymorphism on chromosome 11L between IRBB10 and IR24 and also a great distortion in Xa10inheritance. Rice lines that carry Xa10gene confer race-specific resistance to Xoostrains harboring avirulence (Avr) gene avrXa10. The Xa10locus was roughly integrated to a large region between RFLP markers RG103 (~83 cM) and RG1109 (~91.4 cM) on the long arm of rice chromosome 11 . High-resolution mapping by Gu et al.  narrowed down Xa10to a 74 kb region between RFLP markers M491 and M419. Out of the 7 identified genes in this region, 6 are considered possible candidate genes for Xa10. Ogawa and Yamamoto  identified another BB resistance gene Xa11. Xa11confers specific resistance to strains T7156, T7147, T7133, and H75304 (Japanese Xooraces IB, II, IIIA, and V, respectively). Xa11was mapped between the loci of RM347 (2.0 cM) and KUX11 (1.0 cM) on the long arm of chromosome 3 . It was reported that a single dominant gene, Xa12, controlled the resistance in Kogyoku and Java14 to BB race V of Indonesia .
The xa13gene was first discovered in the rice variety BJ1 and this gene specifically confers resistance to the Philippine Xoorace 6, the most virulent race and one not overcome by most reported Rgenes [61–63]. Sanchez et al.  reported the genetic and physical mapping of xa13gene using two F2 populations of the cross between IR24 and IRBB13. The gene was fine mapped to a region less than 4 cM on the long arm of chromosome 8 between two RFLP markers, RG136 and R2027. Sequence analysis of the 14.8 kb fragment carrying xa13gene indicated only two apparently intact candidate genes—an extensin-like gene and a homologue of nodulin (MtN3) and the 5′ end of a predicted hypothetical protein . The expression of the rice gene Os8N3, a member of the MtN3 gene family from plants and animals, was elevated upon infection by Xoostrain PXO99A and depended on type III effector gene PbXo-1. Os8N3resides near xa13and could not be induced in rice lines with Xa13. Inhibitory RNA silencing of Os8N3produced PX099A resistant plants .
Xa14identified from Taichung Native 1 is a dominant gene against resistance to PXO112, BB race 5 of the Philippines . Xa14was located on chromosome 4 between marker RG620 and G282, with 20.1 and 19.1 cM, respectively by Tan et al. . The gene was fine mapped by using two F2 populations. Combining recombination frequencies for the two populations together, the gene Xa14was mapped to 3 BAC clones spanned approximately 300 kb in length between SSR markers HZR970-8 and HZR988-1 .
A series of nine genes (xa15, Xa16, Xa17, Xa18, xa19, xa20, xa26, Xa27, and xa28) have been obtained from mutagenesis which had different resistance levels and resistance spectrums [58, 68–73]. A mutant rice line, XM5, was resistant to bacterial blight . Allelic relationship studies with the known recessive genes, xa5, xa8, and xa13revealed that the gene was not allelic to them and was a new gene. It was designated as xa19.
Devadath  identified a strain of Oryza barthiiwhich was resistant to all the races of Xooin India. Khush et al.  multiplied the strain at IRRI and studied its resistance to Philippine Xooraces. It was found that the strain was akin to Oryza longistaminatawhich was resistant to all the six Philippine Xooraces . F1s of the cross of the strain with IR24 were resistant showing that the resistance was conferred by a dominant gene. The gene was different from the known BB resistance genes and was designated as Xa21(t). Genetic and physical analysis of Xa21localized it in an 8.3 cM interval on chromosome 11 and the physical size of the region containing Xa21gene was estimated to be about 800 kb .
Genomic DNA gel-blot analysis revealed that Xa21belonged to a multigene family containing at least eight members. Most of these members were mapped to a single locus on chromosome 11, which is linked to at least nine other major resistance genes and one QTL for resistance [7, 77]. Wang et al.  studied the inheritance patterns and resistance spectra of transgenic plants carrying six Xa21gene family members and observed that one member, designated Xa21D, conferred only partial resistance. Analysis of Xa21Dsequence showed that Xa21Dtranscript terminated shortly after the stop codon introduced by retrotransposon. Sequence comparison of Xa21and Xa21Dprovided evidence of adaptive selection. Xa21Dencodes a receptor-like protein carrying LRR motifs in the presumed extracellular domain. Both functional and evolutionary evidence indicated that Xa21DLRR domain controlled race-specific pathogen interaction.
Another BB resistance gene, Xa22was identified from landrace Zhachanglong (ZCL) of Yunnan province in Southwest China. It showed high level of resistance to 16 of the 17 BB strains tested . The gene was nonallelic to Xa1, Xa2, Xa4, and Xa14but was found to be linked to Xa4. Xa22gene was mapped on short arm of chromosome 11, but according to new molecular map developed by Harushima et al. , it was present on the long arm. Xa22provides resistance to a broad spectrum of Xooisolates. The gene was localized to a small 100 kb fragment delimited by R1506 and a subclone from M3H8 BAC clone by a combination of genetic recombination analysis and physical mapping .
An interspecific cross was made between RBB16 and Jiagang30 (JG30), and the F1 plants showing highest resistance to BB were anther cultured to obtain doubled haploids for resistance. These anther-cultured progenies were inoculated with three strains of Xoo, and lines showing high resistance and improved plant type in H2 generation were advanced to H4 and collectively designated as WBB1. Zhang et al.  crossed WBB1 with two susceptible Chinese varieties, JG30 (indica) and 02428 (japonica), and IRBB21 (Xa21). Inoculation results with PXO99 at seedling stage suggested that a single dominant gene from Oryza rufipogonconferred resistance and the gene was nonallelic but probably linked to Xa21. PCR analysis of WBB1 with Xa21specific primers also indicated that the gene was different from Xa21. The new gene, designated as Xa23(t), was mapped within a 0.4 cM region between markers Lj138 and A83B4, and the corresponding physical distance between these markers is 49.8 kb. Six Xa23candidate genes have been annotated, including four candidate genes encoding hypothetical proteins, and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. Nine varieties (AC19-1-1, Aus 274, Chinsurah Boro II, Kalimekri 77-5, Tepa I, Long grain, Aus 295, DV85, and DV86) were crossed with BJ1 (carrying xa13) to confirm whether the resistance was due to xa13or a new gene. The results indicated that the recessive gene identified in DV86 for resistance to race 6 was nonallelic to and independent of xa13. DV85, DV86, and Aus 295 had the same recessive resistance gene and it was designated as xa24(t) .
The xa24resistance gene was fine mapped to a 71 kb DNA fragment in the long arm of chromosome 2 using PCR-based markers. It mediates resistance to the Philippine Xooraces 4, 6, and 10, and Chinese Xoostrains Zhe 173, JL691, and KS-1-21. The analysis of F2 mapping population, developed from the cross between a resistance line DV86 (Oryza sativassp. indica) carrying the target gene and a sensitive line IR24 (O. sativassp. indica), first mapped the gene to terminal region of long arm of chromosome 2 at 8.0 and 0.9 cM from RM482 and RM138, respectively. Further, fine mapping placed the xa24gene between RM14222 and RM 14226 at a distance of 0.07 cM from both markers .
Xa25(t), a new dominant resistance gene, was identified in Minghui 63, a restorer line for a number of rice hybrids cultivated widely in China . The gene conferred resistance to Philippine race 9 (PXO330) at both seedling and adult plant stages. Gao et al.  identified another dominant gene in a somaclonal mutant HX-3 providing resistance to BB strain Zhe173 and was also designated as Xa25(t). Bulked segregant analysis carried out on the DH population located the gene at the terminal region of the long arm of chromosome 4 between the two SSR markers RM6748 and RM1153 at a map distance of 9.3 and 3.0 cM, respectively.
In order to broaden the germplasm pool for breeding of disease resistance to Xoo, a wide hybridization project to transfer Rgenes from wild species Oryza minutawas initiated at IRRI in the late 1980s. A novel resistance (R) locus was identified from a progeny of interspecific hybrids of O. sativacv. IR31917-45-3-2 and O. minutaacc. 101141 and was tentatively designated as Xa27(t) . The genetic analysis of 21 rice cultivars for BB resistance divided the cultivars into two groups (group 1 and group 2) based on their resistance to Philippine Xooraces 1 and 2. Group 1 was resistant to race 1 and group 2 to race 2. When crossed with TN1 and evaluated for resistance with races 1 and 2, all cultivars showed monogenic inheritance of resistance. Allelic relationships of the genes were investigated by crossing these cultivars with different testers having single genes for resistance. Out of all the cultivars, three had new, undescribed genes. Nep Bha Bong had a recessive gene for moderate resistance to races 1, 2, and 3 and resistance to race 5 and was also designated as xa26(t). Arai Raj had a dominant gene for resistance to race 2 which segregated independent of Xa11and was designated Xa27(t). Lota Sail had a recessive gene for resistance to race 2 which segregated independently of Xa10and was designated as xa28(t) .
Gu et al.  reported the fine genetic mapping of the Xa27(t) gene by performing disease evaluation of an Xa27(t) NIL, IRBB27, with 35 Xoostrains from 11 countries. Resistance of Xa27(t) gene was developmentally regulated in IRBB27 and showed semidominant or dosage effect in the cultivar CO39 genetic background. Three markers, M336, M1081, and M1059, were closely linked to Xa27(t). The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1059 markers on rice genome. Markers derived from the genomic sequence of O. sativacv. Nipponbare finally located the Xa27(t) gene within a genetic interval of 0.052 cM on the long arm of chromosome 6 between markers M964 and M1197, and co-segregated with M631, M1230, and M449.
Another dominant Xagene was identified from a rice line ‘B5’ derived from Oryza officinalisthrough introgression. It proved to be highly resistant to brown plant hopper, white-backed plant hopper and bacterial blight. The resistance gene identified in B5 was designated as Xa29(t) . Bulked segregant analysis of RILs from a cross between 'B5' and 'Minghui63' located the resistance gene within a 1.3 cM region flanked by RFLP markers C904 and R596 on chromosome 1.
A new rice BB resistance germplasm (Y238) from the wild rice species O. rufipogonwas identified and designated as Xa30(t) . The gene was mapped on the long arm of rice chromosome 11. Linkage analysis revealed that four molecular markers RM1341, V88, C189, and 03STS were located on the same side of Xa30(t), with genetic distances of 11.4, 11.4, 4.4, and 2.0 cM to the candidate gene, respectively. Wang et al.  identified Xa31(t) gene for BB resistance in Zhachanglong (ZCL), a regional rice variety from Yunnan province in southwest China, which has a high level of resistance to a broad spectrum of Xooisolates. Genetic linkage analysis and fine mapping localized Xa31(t) within a genetic distance of 0.2 cM between two RFLP markers G235 and C600 on the end of long arm of chromosome 4. The flanking markers were used to screen the MH63 BAC library and the Xa31(t) locus was limited to one BAC clone with a length of about 100 kb.
Another novel BB resistance gene from a wild rice (Oryza australiensis) introgression line, C4064, was found to be resistant to Xoostrains PXO61, PXO71, PXO99, PXO145, PXO280, PXO339, and KX085 but susceptible to PXO8 and PXO79. The gene was tentatively designated as Xa32(t). The chromosomal position of the gene was located using BSA with SSR and EST markers between two markers at the end side of long arm and four markers on centromere side. Finally, the gene was mapped within a 2.0 cM interval flanked by two SSR markers RM2064 and RM6293 on the long arm of chromosome 11 . Another BB resistance gene, xa33(t) was identified from rice cultivar ‘Ba7’. The gene was localized on long arm of chromosome 6, where two other dominant genes (Xa7and Xa27) have been reported. RM 20590 was identified as the closest linked marker. Although Xa7and xa33shared common markers, both showed different gene actions and were not growth stage-dependent genes . Natarajkumar et al.  placed a new BB resistance gene, also designated as Xa33(t) from Oryza nivara, on chromosome 7 flanked by the markers RM21004 and RM21177 at a genetic distance of 2.0 and 4.5 cM, respectively.
Pin Kaset (PK), a Thai rice cultivar, had a high level of resistance to BB. The resistance gene in PK was identified using BC2F2 plants from a cross between Ba7 and PK. Phenotypic evaluation of TB0304 and genotypic analysis with SSR markers revealed that the gene was linked to RM224 on chromosome 11 . The new gene was tentatively designated Xa34(t). Although Xa34(t) and other BB resistance genes are located in the same region and shared common linked markers, no evidence was obtained whether they shared the same genomic sequence or were tightly linked to each other. Also, Ram et al.  screened 2 introgression lines (IR65483-118-25-31-7-1-5-B and IR65483-141-2-4-4-2-5-B) derived from IR56 X O. brachyantha, against a virulent isolate DXO44 and 21 different virulent isolates of BB from 11 states of India. The ILs showed resistance to 16 isolates, moderate resistance to one and susceptibility to 4 isolates, as against the differential reaction of IRBB lines to different isolates. This indicated that the introgressed gene is different from those in IRBB lines. It was designated as Xa34(t). Crosses of ILs with IR56 and susceptibility check BPT5204 gave all resistant F1s and 3:1 ratio in F2 revealing a single dominant gene in both cases. Allelic test was done by crossing ILs with IRBB21 which showed that Xa34(t) (resistance gene from O. brachyantha) and Xa21were nonallelic and inherited independently.
A new rice bacterial blight resistance gene Xa35(t) from the wild rice species Oryza minuta(Acc. No. IRGC101133) was identified and transferred into IR24 . Through genetic analysis and identification of resistance spectrum, Xa35(t) showed a high level of resistance to PXO61, PXO112, and PXO339, but was susceptible to PXO86 and PXO99. The Xa35(t) locus was mapped to a 1.80 cM region and this locus was co-segregated with marker RM144, and was 0.7 cM from marker RM6293 on one side and 1.1 cM from marker RM7654 on the other side of the rice chromosome 11. Miao et al.  identified that the rice germplasm C4059 harbored a bacterial blight resistance gene and designated it as Xa36(t). The gene Xa36(t) was mapped on the long arm of rice chromosome 11 encompassing 4.5 cM region flanked by RM224 and RM2136.
An accession of O. nivara(IRGC 81825) was identified to be resistant to all the seven Xoopathotypes prevalent in North India . The F2 population derived from a cross between PR114 and O. nivaraacc. 81825 segregated in a 3:1 ratio of resistant: susceptible plants, indicating that the resistance was provided by a single dominant gene. The inheritance and mapping studies using F2, BC2F2, BC3F1, and BC3F2 progenies of the cross O. sativacv. PR114 × O. nivaraacc. 81825 mapped the gene on chromosome 4L, spanning an approximate 38.4 kb region . Since none of the known Xagenes mapped on chromosome 4L were effective against the Xoopathotypes tested, the gene identified and transferred from O. nivarawas considered novel and designated as Xa38.
A rice introgression line (IL), FF329, identified from a BC1F4 population derived from the cross between donor PSBRC66 (P66) and recipient Huang-Hua-Zhan (HHZ), exhibited a typical hypersensitive response (HR) with all 21 representative Xoostrains (14 Philippines races and seven Chinese pathotypes) . By contrast, the parents were highly susceptible to 10 of the tested strains and resistant or moderately susceptible to 11 strains, but without HR symptoms. As FF329 showed broad-spectrum BB resistance, the gene identified was novel and was designated Xa39. Two SSR markers, RM21 and RM206, located on rice chromosome 11, were linked to the target gene by bulked segregant analysis of the F2 population derived from the HHZ/FF329 cross. The fine mapping of Xa39locus placed the gene in the region of 97 · 4 kb interval flanked by markers RM26985 and DM13.
The japonicaadvanced backcross breeding lines derived from the indicaline IR65482-7-216-1-2 in the background of cultivar Junam was resistant to all Korean BB races. Kim et al.  using two F2 populations derived from the crosses between 11325 (IR83261-3-7-23-6-2-1-1-2-1-2)/Anmi and 11325/Ilpum indicated that resistance (R) was controlled by a new resistance gene designated as Xa40(t), which was co-segregated with the markers RM27320 and ID55. Thus, based on the physical map of japonicarice Nipponbare, the Xa40(t)gene was defined by RM27320 and ID55.WA18-5 located on the BAC clone OSJNBa0036K13 spanning approximately 80 kb region on chromosome 11.
Hutin et al.  screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility “S” gene OsSWEET14, which encodes a sugar transporter targeted by numerous strains of Xoo. They identified a single allele with a deletion of 18 bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. Further, they showed that this allele, which was designated as xa41(t), confers resistance against half of the tested Xoostrains, representative of various geographic origins and genetic lineages, highlighting the selective pressure on the pathogen to accommodate OsSWEET14 polymorphism. Analysis of xa41(t) demonstrated that the resistance through TAL effector-dependent loss of S-gene expression can be greatly fostered upon knowledge-based molecular screening of a large collection of host plants.
A new mutant named ‘XM14’ was obtained by treating IR24, which was resistant to all Japanese Xooraces. The gene identified in XM14 was designated as xa42. The F₂ population from XM14 × IR24 clearly showed 1 resistant:3 susceptible segregation, suggesting control of resistance by a recessive gene. The xa42is located around the centromeric region of rice chromosome 3 in the chromosomal region encompassed by KGC3_16.1 and RM15189.
3. Determinants of pathogenicity
Xanthomonas oryzaepv. oryzae(Xoo) is a member of the c-subdivision of the Gram-negative proteobacteria. It continues to grow in the vascular system until the xylem vessels are clogged with bacterial cells and extracellular polysaccharide (EPS or xanthan). There are several races of Xoo, all of which secrete race-specific effectors into the xylem to trigger individualized response and cause infection. The bacteria also release factors which bind and activate transcription of genes that activate resistance response, known as resistance genes (Rgenes) . The factors that activate Xooresistance genes are known as avirulence factors that determine host specificity via gene-for-gene interactions, reducing the virulence of the pathogen as they are recognized by the host. Since each race of Xooproduces unique virulence and avirulence factors, Rgenes have evolved to provide resistance to individual races of Xoo. It has been known that the interactions of Xoowith plants are determined by hypersensitive response and pathogenicity (hrp) genes, which are required for pathogenicity in susceptible host plants and for the hypersensitive response (HR) in resistant and nonhost plants [3, 100]. A considerable effort has been made to identify genes involved in the pathogenesis of Xooand to understand the roles of the gene products in the disease process. The knowledge of pathogenesis genes has become more clear and distinct from the genome sequence of Xoostrains KACC10331, MAFF311018, and PXO99A [4, 101, 102].
3.1. Avirulence genes
Members of the AvrBs3/PthAfamily of transcription activator-like effectors play a major role in the virulence of X. oryzaepv. oryzae. The avrgene family consists of a repeat sequence in the central domain of the encoded protein with each repeat of 34 amino acids. The number of repeats in different gene families varies, but the amino acids in each repeat are conserved except amino acids at positions 12 and 13, which are referred to as variable regions. The arrangements of the variable regions of all characterized family members appear to be a critical feature for the race specificity of the proteins. The carboxyl terminus of AvrXa7and AvrXa10contains a domain that is structurally similar to the acidic activation domain of many eukaryotic transcription factors, in addition to three nuclear localization signal (NLS) sequences as with AvrBs3family members. It has been postulated that, two raxPand raxQgenes of Xooare required for the activation of AvrXa21. Both raxPand raxQresides in a genomic cluster of sulfur assimilation genes, which encodes an ATP sulfurylase and adenosine-5′-phosphosulfate (APS) kinase. These enzymes function together to produce activated forms of sulfate, APS, and 3′-phosphoadenosine-5′-phosphosulfate (PAPS). It has also been observed that the transcription activation domain and the NLS sequences in AvrXa7are required for the virulence activity, suggesting that the avrgene products act as virulence factors that enter the host nucleus and directly affect host gene transcription . Adaptation of an avirulent bacterial race to host varieties containing a single dominant Rgene often results from the loss of function of the corresponding avrgene. However, the fitness penalty associated with loss of the avrgene may prevent disease epidemics .
3.2. Type III effectors
The interaction of many Gram-negative plant and animal pathogenic bacteria with their hosts depends on a conserved type III protein secretion system (TTSS). The TTSS is encoded by hrpgenes for eliciting either HR on nonhost/resistant host plants or pathogenesis on susceptible hosts. The hrpgene-encoded TTSSs are responsible for translocation of avirulence proteins into the host plant cells. In xanthomonads, the hrpgene cluster comprises six operons (hrpAto hrpF) and is positively regulated by HrpGand HrpX[105–107], repressed in nutrient-rich media, but induced in nutrient-limited media and inside the host [108–111]. HrpG, which is predicted to be a member of the OmpRresponse regulator family of two-component signal transduction systems, regulates the expression of hrpX, encoding an AraC-type transcriptional activator, which then activates the expression of other hrpoperons. The highly conserved hrpgenes named hrcencode the proteins of the apparatus of the type III secretion system, and are critical for pathogenicity and the initiation of disease [100, 112, 113]. A hrpgene cluster was identified in the Xoogenome harboring 26 genes inclusive of hpa2and hrpF. The Xoo hrp PAI(31.3 kb) was larger due to the presence of four transposase genes (about 6 kb) located between hpaBand hrpFgenes than its counterparts of Xac(25.6 kb) and Xcc(23.1 kb). Strong amino acid identity was observed between several orthologous hrpgenes of Xooand Xanthomonas axonopodispv. citri(Xac), as hpaF, hpaP, hrpD5, and hpaAhad identity of 74, 76, 79, and 82% respectively, whereas hrpF(68%), hpa1(65%), hrpB5(66%), and hrpB7(65%) exhibited relatively low similarity. The products of hrpFand hpa1are predicted to be exposed or secreted components of the type III secretion system, which is responsible for contributing to their diversity due to distinct selective pressures in the different hosts. It has been demonstrated that the transcriptional regulator for hrp(trh) and phoPgenes in Xoopositively regulate expression of hrpG, but not known whether the regulation is direct or indirect [114, 115]. The trhgene encodes a putative transcriptional regulator  and phoPencodes a putative response regulator of two-component regulatory systems . It has not been reported whether trhand phoPinfluence the expression of other XrvAtargets such as gumand rpf. It will be of interest to further study the regulation of hrpGexpression by deciphering the functional relationship (if any) between xrvA, trh, and phoPto better understand hrpregulatory mechanisms. A homolog to hrpW, a proposed pectate lyase, was not readily apparent in the Xoogenome but several candidate pectate lyase genes were identified that could function similarly to hrpW.
A review classified all known and candidate TTSS effectors from strains of Xanthomonasspp. into 39 groups based on sequence and structural differences, and similarities . A class of T3 effectors called transcription activator-like effectors (TALE) was first identified due to their relatedness to the T3 effector AvrBs3from Xanthomonas campestrispv. vesicatoria. Some of these TALEs have avirulence activity and three different TAL effectors avrXa7, avrXa10, and avrXa27from Xoohave been cloned [13, 118]. All TALEs have nuclear localization signals (NLS) and an activation domain rich in acidic amino acids (AAD) at their C terminus. Each TALE also contains a central region of multiple 34- to 35-amino acid direct repeats that are nearly identical except the 12th and 13th amino acid residues (so-called repeat variable diaminoacids, or RVD) . The combination of repeat number and composition of RVDs of individual TALEs determine the specificity of the targeted genes [119, 120]. Five additional T3 effectors from Xoohave known contributions to virulence under the appropriate conditions. These genes are pthXo1, pthXo2, pthXo3, pthXo6, and pthXo7. PthXo1is the major TAL effector in many strains, including the common laboratory strain PXO99A, and induces the expression of host gene Os8N3, a member of nodulin 3(N3) gene family and encodes a predicted membrane protein . In addition to PthXo1, two other TAL effectors of PXO99A also contribute to virulence by inducing the expression of two different host genes . PthXo6and PthXo7elevate the transcription of host genes OsTFX1and OsTFIIAγ1, respectively. OsTFX1is a member of bZIP family of transcription factors, which are involved in the regulation of many developmental and physiological processes. Another TAL effector gene, named pthXo8, has been identified in PXO99A with quantitative effects similar to pthXo6. Preliminary evidence indicates that the effector is involved in manipulation of the small RNA pathways of the host.
By using custom-engineered TALEs to investigate the functionality of host target genes involved in Xoo/rice interaction, it has been demonstrated that xa13can be induced by Xooand confer disease susceptibility, lending further evidence for Os8N3(OsSWEET11or Xa13) as an S gene. This approach facilitated to identify another SWEETgene (OsSWEET12) that can act as an S gene, provided that the Xoopathogen contains a corresponding TALE. Further studies revealed that the xa27allele can be activated and triggers resistance to the bacterium expressing a corresponding dTALE. Rice genome contains at least 21 SWEET(or N3) genes with a phylogenetic clade that harbors two known S genes (OsSWEET11and OsSWEET14) and three additional uncharacterized SWEETs[122, 123] generated seven artificial or designer TAL effectors based on the promoter sequences of six loci in rice and transformed them into Xoo. Gene expression analyses indicated all dTALEs were active in trans-activating target genes in rice achieving a 100% success rate in engineering active dTALEs. Gene activation of three alleles (xa27, xa13, and OsSWEET12) led to phenotypic changes in disease resistance or susceptibility in response to Xooinfection, suggesting the feasibility of this approach to the gene functional analysis.
The genomic sequences are available for three strains of Xoo[4, 101, 102]. A comparison of the repetitive regions of PXO99A and MAFF311018 indicates a high degree of rearrangements and shuffling of the genes at all of the loci, to the point where only three genes of 17 in MAFF311018 are identical . The genome structure of XooMAFF 311018 was characterized by large numbers of effector (avr) genes of the avrBs3/pthfamily and insertion sequences (ISs). The high degree of genetic diversity and race differentiation characteristic of this pathogen is due to the presence of mobile elements, which leads to genome inversions and rearrangements. The large numbers of TAL effector genes in these species may reflect the evolutionary “investment” the strains have in utilizing the TAL effectors for virulence. The maintenance of high gene numbers may even be exacerbated by rice breeding and Rgene deployment by farmers over the millennia. It could be inferred that Xootargets different host genes to alter the host physiology and different TAL effectors to have qualitatively different effects on host susceptibility.
3.3. Type II secretion system
The bacterial type II secretion system mediates a two-step process. The proteins that are secreted through this system carry a secretion signal at their N termini and are transported into the periplasmic space through the inner membrane by either the general secretion pathway (GSP) or the twin arginine pathway (TWP) [124, 125]. Transport across the outer membrane is facilitated by the proteins of main terminal branch (MTB) of general secretion pathway (GSP). The Xoogenome encodes a single type II secretion system in contrast to two different type II systems in Xacand Xcc. Mutations in the gene cluster xps, which is required for a functional type II system, result in strains defective in virulence . Type II secreted proteins are mainly toxins and enzymes that target different components of the host cell, and some of these enzymes, including xylanase, cellulase, cysteine protease, cellobiosidase, and lipases, have been characterized as contributors to Xoovirulence. Experimental evidences indicate that rice plants perceive some type II secreted proteins and respond by hypersensitive responses (HRs), and these responses are suppressed by type III secreted effectors . Other factors that contribute to the virulence of Xooinclude EPS, the type II general secretion system and its secreted proteins, and regulation involving genes within the rpfcluster [3, 128–132]. The rpfB, rpfC, rpfF, and rpfGgenes (regulation of pathogenicity factor) of Xoo have been shown to affect virulence in rice, EPS production, xylanase production, and motility [130–132]. The virulence of Xanthomonasalso depends upon cell-to-cell signaling mediated by diffusible signal factor (DSF). Two of the genes in this cluster rpfBand rpfFare involved in the synthesis of DSF. Knockout studies on rpfFindicate the role of DSF in the regulation of levels of extracellular enzymes and EPS . The RpfC/RpfGtwo-component system couples the DSF sensing to intracellular regulatory networks through a second messenger, cyclic diGMP, and a global regulator, Clp. Protein-protein interaction between the DSF synthase RpfFand the sensor RpfCmay act as a posttranslational mechanism to modulate the biosynthesis of DSF [134, 135]. Recently, an X. oryzaepv. oryzaeflagellar operon region has been isolated from Xoo, which contains four ORFs. One of the ORFs, flhF, encodes a putative GTP-binding protein which is involved in chemotaxis. Mutation in flhFresulted in weak chemotaxis but did not show reduced virulence if inoculated on rice leaves with a scissors-clipping method, suggesting that chemotaxis is not required for virulence once the bacterial cells enter rice leaves . EPS synthesis is directed by genes at multiple chromosomal loci; one of the loci is called the gumcluster . The Xoo gumcluster in strain KACC10331 is composed of 14 ORFs arranged in a tandem array, expressed from a promoter located upstream of gumB, but internal promoters can also be found upstream of gumG, gumH, and gumM, respectively [101, 138]. Two different Xoomutants, one with a transposon insertion in gum Gand another with a spontaneous mutation due to the insertion of endogenous IS element in gum M, were incapable of EPS production and less virulent. In Xoo, EPS synthesis has been found to be controlled by the rpfCgene, which is part of a two-component system. Strains carrying a mutation in rpfChave greatly reduced EPS production and virulence but still attain maximum population levels in rice plants  indicating that EPS is a virulence determinant in X. oryzaepv. oryzae.
Recently, screening of a transposon mutant library of a Korean Xoostrain, KACC10331, in rice also showed that Tn5 insertion in the xrvAgene (XOO2744) led to reduced virulence; however, the mutant was not characterized in further detail . The deduced protein encoded by xrvApossesses an H-NS domain. H-NS and H-NS–like proteins are modular proteins associated with the bacterial nucleoid. The discovery of xrvAas a regulator of virulence factor synthesis came from work aimed at identification of genes involved in EPS production of Xoo. Disruption of xrvAled to a significant reduction in virulence, a delay in HR elicitation, a decrease in EPS and DSF production, and an increase in glycogen accumulation.
3.4. Type I secretion systems
Type I secretion systems of Gram-negative bacteria are secretion systems that transport proteins directly to the extracellular environment from the bacterial cytoplasm through inner and outer bacterial membranes. Three highly conserved components of type I secretion systems are an ABC transporter, which forms a channel across the inner membrane, a membrane fusion protein (MFP), and an outer membrane protein called To1C . PXO99A contains a type I secretion system that is involved in triggering resistance in rice cultivars which carry the Xa21resistance gene. Transposon-induced mutations in PXO99A and subsequent screening for mutants that lost Xa21-mediated avirulence activity identified eight genes viz. raxA, raxB, raxC, raxST, raxQ, raxP, raxH, and raxR. AvrXa21activity requires the presence of raxA, raxB, and raxCgenes, and these three genes encode the MFP, ABC transporter, and outer membrane protein. The AvrXa21pathogen-associated molecule is involved in quorum sensing and the expression of raxSTis regulated by a two-component regulatory system encoded by raxHand raxR that responds to Xoocell population density, and may be conserved in most Xanthomonasspp. The raxSTencodes a sulphotransferase enzyme, while raxQand raxPare involved in the production of the sulfuryl donor phosphoadenosine phosphor sulfate (PAPS). The elicitor of Xa21immunity, Ax21, was characterized as a 194 amino acid sulfated protein, which is secreted into the extracellular environment . The N-terminal 17 amino acid peptide of Ax21is sulfated at a tyrosine residue and is sufficient to trigger Xa21-mediated resistance.
4. Molecular mechanism of BB resistance in rice
Out of 42 Xagenes identified so far, nine genes (Xa1, Xa3/Xa26, xa5, Xa10 xa13, Xa21, Xa23, xa25, and Xa27) have been characterized at molecular level and these encode various types of proteins (Table 2). Based on these studies, the molecular mechanisms of BB resistance in rice seem to be largely different from the mechanisms of resistance to rice blast, although the mechanisms of rice disease resistance remain largely to be elucidated. Most of the characterized BB resistance genes are different from the most common R protein, nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein . Interestingly, except for the fact that Xa21 and Xa26 encode for similar receptor-like proteins, the products of the other genes are unique and not found in other plant species . These features suggest that molecular mechanism of rice-Xoosystem is more complicated and a unique pathosystem to study the interactions between hosts and pathogens. The molecular mechanism of BB resistance gene has also been discussed in other book chapters [146, 147]. The nine characterized BB resistance genes fall into six different classes of proteins and thus may give a wide scenario of understanding at molecular level.
|S. No.||Gene||Encoded protein||Reference|
|2||Xa3/Xa26||Leucine-rich repeat receptor-like kinase (LRR-RLK)|||
|3||xa5||TFIIA Transcription factor||[11, 12]|
|4||Xa10||Executor R protein, encodes 126 AA, with four potential transmembrane helices|||
|7||Xa23||Executor R protein, encodes 113 AA, with four potential transmembrane helices|||
|9||Xa27||Apoplast (rice unique gene)|||
The BB resistance genes characterized at molecular level.
4.1. BB resistance conferred by LRR receptor kinase protein
The LRR receptor kinase class of BB resistance is conferred by Xa21and Xa3/Xa26genes. The Xa21was the first rice BB resistance gene characterized  and was one of the most intensively studied genes at molecular level. It was originally identified in the wild species O. longistaminata, and isolated by map-based cloning strategy from the IRBB21, a near-isogenic line in the background of IR24. This gene confers a race-specific resistance to Xoo, and is the most widespread BB resistance gene in the rice cultivated area, thereby providing broad-spectrum resistance. The receptor kinase-like protein encoded by Xa21consists of putative extracellular domain containing LRR, a single pass transmembrane domain, and an intracellular domain containing serine/threonine kinase. This protein is unique in carrying the receptor domain LRR with hypothetical function in pathogen recognition, and the kinase domain that functions in subsequent signal transduction as compared to other cloned plant resistance genes . The Xa21-mediated resistance is not expressed in the early developmental stages and gradually increases from the seedling stage to later stages, with 100% resistance at the adult stage . The gradual increase in expression of Xa21gene during rice development is associated with development-controlled Xa21-mediated resistance . Ectopic expression of Xa21gene can generate rice plants with a high level of resistance to Xooat both seedling and adult stages [150, 151]. The Ax21(avirXa21protein; as called activator of Xa21) protein was secreted by Xoothrough its type I secretion system switch on the Xa21gene present in the host . A sulfated 17-amino acid synthetic peptide derived from the N-terminal region of Ax21is sufficient for its initiation. The Ax21is highly conserved in many Xanthomonasspecies including number of pathogens of plants and human across microbial genus. Thus, Ax21is considered a pathogen-associated molecular pattern, and thus, Xa21can be classified both as a plant pattern recognition receptor (PRR) and an R protein .
The Xa21 locus is a multigene family and several key components of Xa21protein such as E3 ubiquitin ligase/XB3, WRKY62/XB10, protein phosphatase 2C (PP2C)/XB15, ATPase/XB24, and Bip3 (also known as glucose-regulated protein), involved in defense signaling pathway have been identified. The E3 ubiquitin ligase interacts with the kinase domain of Xa21protein and acts as a substrate for the Xa21serine and threonine kinase activity, which is necessary for full accumulation of the Xa21 protein, thus Xa21-mediated immunity . The Xa21protein binds to WRKY62 with catalytic activity of its juxtamembrane motif and serine/threonine kinase domain. The WRKY62 and WRKY76 (another WRKY transcription factor) function as a negative regulator of Xa21-mediated resistance [153, 154]. The PP2C component interacts with juxtamembrane motif and kinase domain of Xa21protein, and can dephosphorylate autophosphorylated Xa21, thus acting as negative regulator of Xa21-mediated resistance . The XB24, an Xa21binding protein, was isolated using yeast two-hybrid screening and belongs to a class of ATPases. The ATPase/XB24 can enhance autophosphorylation of Xa21protein by its physical association in vivowith the juxtamembrane motif and kinase domain, and downregulates the Xa2-mediated resistance Xa21protein by its enzymatic activity, because the activation of Xa21following interaction with pathogen-associated molecular pattern Ax21requires the dissociation of XB24 from Xa21or removal of autophosphorylation. The rice plants with reduced level of XB24 expression showed enhanced level of Xa21-mediated resistance, whereas Xa21-mediated resistance overexpressing the XB24/ATPase in rice plants showed lower level of Xa21-meditated resistance, as XB24 in this case cannot readily dissociate from Xa21and further binding of Ax21 with Xa21leads to conformational change in Xa21protein. The conformational change in Xa21protein exposes it to degradation in endogenous proteases leading to lower resistance 21 protein a21. The endoplasmic reticulum chaperone Bip3 can interact with Xa21protein in vivo. Rice plants overexpressing Bip3 have decreased Xa21protein accumulation and inhibited Xa21protein processing, which results in compromised Xa21-mediated resistance .
The Xa3/Xa26 locus was isolated as Xa26from an indicarice cultivar Minghui 63  and found to be similar to previously identified gene Xa3, therefore renamed as Xa3/Xa26. It encodes a plasma membrane-localized LRR receptor kinase-type protein with an extracellular LRR domain, a transmembrane motif, and a cytoplasmic kinase domain . It also confers a broad-spectrum resistance relative to Xa21and has been widely deployed in rice cultivars in China [158–160]. Two different alleles of Xa3/Xa26have been identified from “CC” genome of O. officinalisand O. minutathat can mediate a similar spectrum of resistance against Xoo. This indicated the early origin and relatively conserved function of Xa3/Xa26 locus during evolution that can provide durable resistance against Xoo. The MRKa gene, an ortholog of Xa3/Xa26 gene family in rice cultivar Mingui 63, can mediate partial resistance to Xoowhen it is overexpressed . The kinase domain is important for complete function of Xa3/Xa26 for resistance, as rice plants carrying truncated Xa3/Xa26 gene without kinase domain exhibits lower level of resistance. The kinase domain of MRKa protein can partially restore the function of the truncated Xa3/Xa26 gene in Xooresistance, suggesting the partially conserved function of the orthologs of this family. This hypothesis is also supported by another study that NRKe gene from rice cultivar Nipponbare gets transcriptionally activated in response to raised temperature . The kinase domain of Xa3/Xa26protein can replace the function of the kinase domain of NRKe protein in response to temperature change.
The genetic background and development stage of rice plant affect the Xa3/Xa26-mediated resistance. The higher level of resistance was observed in japonicaas compared to indicabackground. The Xa3/Xa26expression gradually increases from seedling stage to adult stage and pathogen infection also differentially effects its expression in plants with different genetic backgrounds, Xoostrains at both seedling and adult stages, but has full resistance to other Xoostrains at adult stage [8, 163, 164]. Further resistance mediated by Xa3/Xa26increases with increase in its expression and its constitutive expression provides broad-spectrum resistance at both seedling and adult stage without affecting the agronomic performance .
Several members functioning downstream in the Xa3/Xa26-initiated defense signaling pathway have been identified, where the downstream function of these components can mediate a broad-spectrum resistance as compared to Xa3/Xa26 protein. Two WRKY-type transcription factors namely WRKY13 and WRKY45-2 positively regulate rice resistance to Xoo, of which M. oryzae(causal organism of rice blast), with WRKY13 putatively functions upstream of WRKY45-2 in rice-Xoointeraction [165–167]. Two genes namely OsDR8 encoding a protein involved in thiamine biosynthesis and C3H12, a CCCH-type zinc finger nucleic acid-binding protein, also act as positive regulator of rice resistance to Xooin Xa3/Xa26-initiated defense pathway [152, 168]. Of these, C3H12 functions upstream of WRKY45-2. A rice tribe-specific gene OsDR10 that probably functions upstream of WRKY13, negatively regulates resistance to Xooin Xa3/Xa26-mediated resistance pathway .
Both Xa21 and Xa3/Xa26belong to multigene family; encode same type of proteins and have 53% sequence similarity [7, 8]. The only structural difference between two genes is the number of LRR, where Xa26encodes 26 LRR, whereas Xa21encodes 23 LRR . However, the respective LRR domains of Xa3/Xa26and Xa21are the important determinants of race-specific recognition during rice-Xoointeractions as evidenced from experiment on domain swapping analyses, but a juxtamembrane motif of Xa3/Xa26 also seems to contribute in resistance specificity . The kinase domain of Xa3/Xa26 can partially replace the function of the kinase domain of Xa21, or vice versa, in Xooresistance, suggesting the partially conserved nature of this domain in defense signaling pathway . Both Xa3/Xa26and Xa21genes are dose-dependent and their expression progressively increases with developmental stage. However, Xa3/Xa26has a higher expression level in the japonicabackground than in an indicabackground [150, 164]. Further study may also be required to determine, whether Xa3/Xa26is also a PRR in addition to being an R protein.
4.2. BB resistance conferred by MtN3/saliva class protein
The MtN3 (a homologue of nodulin protein) class of BB resistance is conferred by xa13, a fully recessive gene for BB resistance. The gene encodes a novel protein that has no sequence similarity with any known Rproteins, but it shows 50% sequence identity and 68% sequence similarity to the product of a nodulin MtN3 gene in legumes . The xa13 and its dominant allele Xa13, which is also named Os8N3and OsSWEET11 [64, 122], encode identical polytopic plasma membrane proteins of the MtN3/saliva family proteins, but have crucial sequence differences in their promoter regions. Promoter swapping analysis confirms that Xa13(Os8N3and OsSWEET11) is a susceptibility gene, which is induced by direct binding of the transcription activator-like (TAL) effector PthXo1 of Xoostrain PXO99 to the cis element, the UPT PthXol box, on the Xa13promoter [64, 170, 171]. The Xoostrain PXO99 that secretes PthXo1 cannot induce recessive xa13due to the mutation of the UPT PthXo1 box in the xa13promoter. Further, PXO99 is more sensitive to copper (an essential micronutrient of plants, is also an important element for a number of pesticides in agriculture) than other Xoostrains. The Xa13protein interacts with two copper transporter-type proteins, COPT1 and COPT5, to promote removal of copper from xylem vessels, where Xoomultiplies and spreads to cause disease . As PXO99 cannot induce recessive xa13, the copper levels in rice plants carrying the recessive xa13 gene can inhibit Xoogrowth and thus plants show resistant reaction. Besides functioning against Xooresistance, product of xa13gene also has an essential role in pollen development. This became evident from suppressing the function of either the dominant or recessive allele of xa13in rice transgenic plants, where xa13not only enhanced the resistance but also caused male sterility . The study showing the link between two unrelated biological processes further demands the detailed studies in the future on the functional overlap between pathogen-induced defense signaling and plant development.
The recessive xa25gene, also named Xa25(t), encodes a plasma membrane protein of the MtN3/saliva family similar to xa13and confers race-specific resistance to Philippine Xoostrain PXO339 [19, 83]. The protein encoded by xa25and its dominant allele differ in eight amino acids. The Xoostrain PXO339 can induce the expression of dominant Xa25, but not recessive xa25. The differences in proteins and expression pattern of xa25and its dominant allele Xa25in rice-PXO339 interaction suggest that the dominant Xa25may be a race-specific susceptible gene, whereas the recessive xa25has evolved as the mutant that cannot be induced by rice-Xoointeraction—similar to the recessive xa13. The developmental stage of rice plant also influences the xa25-mediated resistance. The xa25gene regulated by its native promoter, when transferred in the rice plants homozygous for Xa25, behaves in recessive nature. The rice plants homozygous for xa25showed resistant reaction on inoculation with PXO39. However, the rice plants heterozygous at the xa25locus were susceptible to PXO339 at the seedling stage, but became resistant to PXO339 at the adult stage. The dominance reversal characteristic of xa25may be because of suppression of PXO339-induced activation of dominant Xa25 at the adult stage . The rice MtN3/saliva family contains more than 20 paralogs. Some MtN3/saliva proteins from different species act as glucose transporter [122, 174]. The Xa13, besides functioning as a susceptibility gene in race-specific rice-Xoointeraction, also acts as sucrose transporter. Similar role needs to be elucidated for Xa25gene along with rice-Xoointeraction.
4.3. BB resistance conferred by TAL effector-dependent class protein
Three BB resistant genes Xa27, Xa10, and Xa23act as transcription activator-like (TAL) effector-dependent Rgenes [13, 20, 21]. The Xa27mediates race-specific resistance to diverse strains of Xoo, including Chinese and Philippine Xooraces . This gene encodes an apoplast protein of 113 amino acids that has no distinguishable sequence similarity to proteins from organisms other than rice . The resistant and susceptible alleles of Xa27encode an identical protein, but they differ from each other only in the promoter region . The Xa27-mediated race-specific resistance to Xoodepends upon AvrXa27, a TAL effector of Xoothat induces resistance reaction by binding to the UPTAvrXa27 box of the Xa27promoter . However, the recessive MR gene xa5can attenuate the Xa27-mediated resistance in rice, which suggests that XooTAL effector could not use protein encoded by the recessive xa5as a transcription machinery for activation of Xa27. The secondary cell wall thickening in vascular bundle elements is obviously associated with Xa27-mediated resistance . According to the molecular models of plant innate immunity, pathogen-associated molecular pattern-triggered immunity (PTI) is induced by plasma membrane-localized plant pattern recognition receptors (PRRs) and effector-triggered immunity (ETI) is initiated by NBS-LRR–type R proteins present in the cytoplasm [178, 179]. The subcellular localization and sequence specificity of Xa27protein suggest that it does not follow either PTI or ETI procedure , though its typical biochemical function in rice-Xoointeraction still remains to be elucidated.
Xa10, another TAL effector-dependent Rgene for BB resistance contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10expression . The Xa10-encoded protein consists of 126 amino acid residues, which is predicted to have four potential transmembrane helices (M1–M4), among which, M2 contains charged amino acid residues. Xa10is an inducible protein that triggers programmed cell death by disruption of the endoplasmic reticulum (ER) and cellular Ca2+ homeostasis. The Xa10functions not only in rice but also in other species such as Nicotiana benthamiana, and in animal kingdom such as mammalian HeLa cells . The Xa10protein localizes as hexamers in the ER and is associated with ER Ca2+ depletion in plant and HeLa cells. It shows hypersensitive response with faster cell death and shorter lesions in incompatible reactions. The variants of Xa10that abolish programmed cell death and ER Ca2+ depletion in N. benthamianaand HeLa cells, also abolish disease resistance in rice . In one of the experiment, the modified Xa10gene designated as XA10E5was transferred into Nipponbare and 93-11 backgrounds to generate broad-spectrum resistance to most of the BB pathogens. The XA10E5contains five tandemly arranged effector binding elements (EBEs), each responding specifically to a corresponding virulent or avirulent TAL effector. The transgenic lines with XA10E5showed resistance to 27 of the 28 Xoostrains collected from 11 countries .
Xa23, another TAL effector associated executor Rgene, was isolated from O. rufipogonusing map-based cloning and TAL effector-based technology . It encodes 113 amino acid long proteins (same number of amino acids as in Xa27) with 64% similarity only to Xa10protein, but nonsignificant similarity at genomic level. In addition, the predicted three transmembrane helices of Xa23largely overlap with transmembrane helices (M2–M4) of Xa10indicating that Xa23 could be a homolog of Xa10. A paralog of Xa23has been found to exist in Nipponbare genome with unknown function. The AvrXa23 is responsible for activation of Xa23transcription by a TAL effector present in all examined Xooisolates . As compared to Xa10, Xa23is a broad-spectrum Rgene and provides strong resistance to all natural Xoostrains tested so far possibly due to presence of avrXa23 in natural Xoostrains . The susceptible xa23allele Xa23but differs in promoter region by lacking the TALE binding element (EBE) for AvrXa23. Xa23can trigger a strong hypersensitive response in rice, tobacco, and tomato .
4.4. BB resistance conferred by NBS-LRR class proteins
Xa1is the only NBS-LRR–type BB resistance gene identified so far and is an inducible gene that means its expression is induced by wounding and pathogen infection . However, out of number of uncharacterized BB resistance genes, some might be providing resistance through NBS-LRR class of proteins. One such gene is Xa38, identified from O. nivaraseems to be of NBS-LRR type based on the putative candidate gene sequence analysis in the targeted bacterial artificial clone (BAC) . Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes play important roles in plant disease resistance. They are generally believed to be responsible for the recognition of effectors delivered by pathogens during infection and the induction of downstream disease resistance reactions . Plant NBS-LRRs can be divided into several types based on the variable N-terminus. In rice, most NBS-LRRs belong to the CC-NBS-LRR (CNL) subclass, in which there is a coiled-coil domain at the N-terminus [185, 186]. In the genome of rice (O. sativacv. Nipponbare), more than 400 NBS-LRR genes have been identified , but although these genes are believed to be defense-related, the functions of most of them are unknown in rice genome. Recently, a novel NBS-LRR gene was isolated from rice and designated as Oryza sativaRp1-like 1 (OsRP1L1) based on similarity to Rp1 locus of maize, an intensively studied NBS-LRR gene providing resistance to common leaf rust in maize . Overexpression of OsRP1L1 moderately elevated the resistance of plants to Xoostrains PXO86 and PXO341 in a susceptible japonicacultivar . Similarly, a large number of genes out of 400 NBS-LRR in the rice genome can be isolated that could be putative candidates for BB resistance.
4.5. BB resistance conferred by other class of protein
The recessive xa5that belongs to this class encodes a typical gamma subunit of transcription factor IIA (TFIIAγ), which is one of general transcription factors required for transcription by RNA polymerase II . It mediates specific resistance to Japanese races and Philippine races 1, 2, 3, and 5 by restriction of bacterial movement, but not multiplication [11, 189]. This gene was cloned by a map-based cloning approach combined with allele sequence analysis , and further complementation testing confirmed this gene . TFIIAγ is involved in the recruitment of the basal transcription machinery by eukaryotic transcription factors. The xa5allele contains a missense mutation that does not seem to influence its function in the recruitment of the basal transcription machinery . It is speculated that XooTAL effectors usurp parts of plant basal transcription machinery to regulate rice gene expression; the missense mutation of xa5allele does not compromise its general function in transcription, but it may evade TAL virulence functions [119, 177]. The Xoo avrXa5is an avirulence gene, which encodes a TAL-type protein, corresponding to xa5. The xa5showed a constitutive expression pattern in different tissues, and the resistance of xa5is not dose-dependent [11, 12].