Transgenic Rice Plants Harboring Genomic DNA from Zizania latifolia Confer Bacterial Blight Resistance

Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.

Application of Functional Markers to Identify Genes for Bacterial Blight Resistance in Oryza rufipogon

Field resistances of nine accessions of common wild rice （Oryza rufipogon Griff.） and one rice variety （IR24） were evaluated by using nine strains of bacterial blight pathogen （Xanthomonas oryzae pv. oryzae） from the Philippines. IR24 was highly susceptible to all the strains, and six common wild rice accessions resisted all the nine strains, with a resistance frequency of 67%. The accessions Yulin and Wanning were only susceptible to PXO280 and PXO71, respectively. The accession Gaozhou was susceptible to the three strains PXO79, PXO99 and PXO339, whereas resistant to the other six strains. It could be concluded that there is at least one resistance gene in each common wild rice accession. The functional markers of the genes xa5, xa13, Xa21 and Xa27 were used to detect the presence of these resistance genes in the nine tested wild rice accessions, and it was found that four wild rice accessions contained heterozygous xa13. Among the nine common wild rice accessions, five were homozygous for Xa27 and three homozygous for xa27, and the accession Laibin contained neither xa27 nor Xa27. In addition, there were no xa5 and Xa21 in all of these accessions.

Evaluatien of Near-lsogenic Rice Lines with 8 Genes for Bacterial Blight Resistance to Strains in China

A set of near-isogenic rice lines withmonogenic resistance to bacterial blight weredeveloped by IRRI.The Cultivar IR24 wasused as the recurred parent.They wereevaluated with 6 races of Xanthomonascampestris pv.oryzae(Xco)in the Philip-pines at the maximum tillering and the bootingstages by ZHANG and MEW at IRRI in 1989.

Improving the resistance of the rice PTGMS line Feng39S by pyramiding blast,bacterial blight,and brown planthopper resistance genes

Knowledge of rice(Oryza sativa L.)genes and various DNA markers can be used in genomic breeding programs aimed at developing improved elite rice cultivars.We used an efficient genomic breeding approach to pyramid four resistance genes(Pi2,Xa23,Bph14,and Bph15)in the popular photoperiod-and thermosensitive genic male sterile(PTGMS)rice line Feng39S.We performed foreground selection for the target genes,followed by recombinant selection and background selection.This process reduced the sizes of the genomic segments harboring the target genes(566.8 kb for Pi2,1143.9 kb for Xa23,774.7 kb for Bph14,and 1574.9 kb for Bph15)and accelerated the recovery of the recurrent parent genome to proportions ranging from 98.77%to 99.16%,thus resulting in four near-isogenic lines.To assemble the four resistance genes in Feng39S,we performed a double-way cross combined with foreground and background selection to generate two improved lines of Feng39S(Pi2+Xa23+Bph14+Bph15)with a recurrent parent genome recovery of 98.98%.The two lines showed agronomic performance,grain quality,and fertility–sterility transition characteristics similar to those of the original Feng39S line.The newly developed PTGMS lines and corresponding hybrid combinations were resistant to various field blast isolates and seven representative isolates of bacterial blight.At the seedling stage,the lines also showed resistance against brown planthopper.This study provides an efficient and accurate genomic breeding approach for introducing desirable traits into PTGMS lines.

Marker-Assisted Pyramiding of Genes Conferring ResistanceAgainst Bacterial Blight and Blast Diseases into Indian RiceVariety MTU1010

Two major bacterial blight （BB） resistance genes （Xa21 and xa13） and a major gene for blastresistance （Pi54） were introgressed into an Indian rice variety MTU1010 through marker-assistedbackcross breeding. Improved Samba Mahsuri （possessing Xa21 and xa13） and NLR145 （possessingPi54） were used as donor parents. Marker-assisted backcrossing was continued till BC2 generationwherein PCR based functional markers specific for the resistance genes were used for foregroundselection and a set of parental polymorphic microsatellite markers were used for background selectionat each stage of backcrossing. Selected BC2F1 plants from both crosses, having the highest recoveriesof MTU1010 genome （90% and 92%, respectively）, were intercrossed to obtain intercross F1 （ICF1） plants,which were then selfed to generate 880 ICF2 plants possessing different combinations of the BB andblast resistance genes. Among the ICF2 plants, seven triple homozygous plants （xa13xa13Xa21Xa21Pi54Pi54）with recurrent parent genome recovery ranging from 82% to 92% were identified. All the seven ICF2plants showed high resistance against the bacterial blight disease with a lesion lengths of only 0.53–2.28 cm, 1%–5% disease leaf areas and disease scoring values of ‘1’ or ‘3’. The seven ICF2 plants wereselfed to generate ICF3, which were then screened for blast resistance, and all were observed to behighly resistant to the diseases. Several ICF3 lines possessing high level of resistance against BB andblast, coupled with yield, grain quality and plant type on par with MTU1010 were identified and advanced forfurther selection and evaluation.

A substitution mutation in OsPELOTA confers bacterial blight resistance by activating the salicylic acid pathway

We previously reported a spotted-leaf mutant pelota（originally termed HM_（47）） in rice displaying arrested growth and enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae. Here, we report the mapbased cloning of the causal gene OsPELOTA（originally termed spl^（HM47））. We identified a single base substitution from T to A at position 556 in the coding sequence of OsPELOTA, effectively mutating phenylalanine to isoleucine at position 186 in the translated protein sequence. Both functional complementation and over-expression could rescue the spotted-leaf phenotype. OsPELOTA, a paralogue to eukaryotic release factor 1（eRF_1）, shows high sequence similarity to Drosophila Pelota and also localizes to the endoplasmic reticulum and plasma membrane.OsPELOTA is constitutively expressed in roots, leaves,sheaths, stems, and panicles. Elevated levels of salicylic acid and decreased level of jasmonate were detected in the pelota mutant. RNA-seq analysis confirmed that genes responding to salicylic acid were upregulated in the mutant. Our results indicate that the rice PELOTA protein is involved in bacterial leaf blight resistance by activating the salicylic acid metabolic pathway.

Identification of molecular markers linked to rice bacterial blight resistance genes from Oryza meyeriana

Y73 is a progeny of asymmetric somatic hybridization between Oryza sativa cv.Dalixiang and the wild rice species Oryza meyeriana.Inoculation with a range of strains of Xanthomonas oryzae pv.oryzae showed that Y73 had inherited a high level of resistance to rice bacterial blight(BB)from its wild parent.An F2 population of 7125 individuals was constructed from the cross between Y73 and a BB-susceptible cultivar IR24.After testing 615 SSR and STS markers covering the 12 rice chromosomes,186 markers were selected that showed polymorphism between Y73 and IR24.Molecular markers linked to the BB resistance genes in Y73 were scanned using the F2 population and the polymorphic markers.The SSR marker RM128 on chromosome 1,the STS marker R03D159 on chromosome 3 and the STS marker R05D104 on chromosome 5 were found to be linked to the rice BB resistance genes in Y73.

Analyses of gene effects and inheritance of resistance to bacterial blight in rice

We analyzed the resistant inheritance of ajaponica variety,Jia23,to two bacterial blightpathogen strains,KS-6-6 and Zhe 173,rep-resenting respectively the two predominantpathogenic types(Ⅱ and Ⅳ)in the rice crop-ping area along the Yangtze River Valley.Jia23 was crossed with susceptible vari-eties,Ewan8 and 7416.Fplants were back-

Primary study on the resistance to bacterial blight(X. oryzae)in Cecropin B gene transgenic rices

Bacterial blight (BB) is one of the major dis-eases to rice. Antibacterial Cecropin B genehas been cloned and transformed into rice. Westudied the resistance to bacterial blight inCecropin B gene transgenic rices.Rice variety JYll9 transformed withCecropin B gene by particle bombardment andprogenies were randomly planted in the field in

Breeding of near-isogenic japonica rice lines with three major genes for resistance to bacterial blight

From 1986 to 1993, a set of near-isogenic japonicarice Iines with three major genes Xα-3, Xα - 4,and Xα-12 for resistance to bacterial blight(Xan-thomonas oryzae pv.oryzae)were developed anddesignated as CBB3, CBB4, and CBB12 respective-

Inheritance of resistance to bacterial streak and bacterial blight in rice

Bacterial streak (BS) caused by Xanthomonascampestris pv. oryzicola has become one of themajor diseases in southern rice areas. Up todate, there are few reports on the inheritanceof resistance to BS and its relationship with theresistance to bacterial blight (BB). Therefore,we analyzed the inheritance of resistant to BSin three resistant rice cultivars and the geneticrelationship of resistance between BS and BB.

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L.

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.

Identification and Genetic Analysis of a Novel Rice Spotted-Leaf Mutant with Broad-Spectrum Resistance to Xanthomonas oryzae pv. oryzae

A spotted-leaf mutant of rice HM143 was isolated from an EMS-induced IR64 mutant bank. Brown lesions randomly distributed on leaf blades were observed about 3 wk after sowing. The symptom lasted for the whole plant growth duration. Histochemical analysis indicated that cell death occurred in and around the site of necrotic lesions accompanied with accumulation of hydrogen hyperoxide. Agronomic traits were largely similar to the wild type IR64 except seed setting rate and 1 000-grain weight which were significantly decreased in the mutant. Disease resistance of the mutant to multiple races of Xanthomonas oryzae pv. oryzae was significantly enhanced. Genetic analysis showed that the mutation was controlled by a single recessive gene, tentatively termed splHM143. In addition, using molecular markers and 1023 mutant type individuals from an F2 segregating population derived from the cross HM143/R9308, the spotted-leaf gene was finally delimited to an interval of 149 kb between markers XX25 and ID40 on the long arm of chromosome 4. splHM143 is likely a novel rice spotted-leaf gene since no other similar genes have been identified near the chromosomal region.

RICE TRANSFORMATION WITH A PHYTOALEXIN GENE AND BIOASSAY OF THE TRANSGENIC PLANTS

Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.