Genome-Wide Analysis of von Willebrand Factor A Gene Family in Rice for Its Role in Imparting Biotic Stress Resistance with Emphasis on Rice Blast Disease

von Willebrand factor A(vWA)genes are well characterized in humans except for few BONZAI genes,but the vWA genes are least explored in plants.Considering the novelty and vital role of vWA genes,this study aimed at characterization of vWA superfamily in rice.Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes,and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other,indicating gene duplication.In addition to vWA domain,vWA proteins possess other different motifs or domains,such as ubiquitin interacting motif in protein degradation pathway,and RING finger in protein-protein interaction.Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling.The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes,indicating the importance of these genes in rice.Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare.qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9,OsvWA36,OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance.This is the first attempt to characterize vWA gene family in plant species.

Enhanced Ultraviolet-B Radiation Suppresses Magnaporthe oryzae Infection and Alleviates Its Damage to the Photosynthesis of Rice Leaves

In the present study,an indoor potting experiment was conducted to study the effects of enhanced UV-B radiation and Magnaporthe oryzae on the growth,stomatal structure,photosynthesis,and endogenous hormone contents of a traditional rice cultivar Baijiaolaojing in the Yuanyang terraces of Yunnan Province.In addition,the relationships between these parameters and disease indices were analyzed.We aimed to clarify the response of the photosynthetic physiology of rice under the combined stress of UV-B radiation and M.oryzae.Compared with the M.oryzae infection treatment,all the treatments,including M.oryzae infection before(MBR),simultaneously with(MSR),and after(MAR)UV-B radiation significantly increased the rice height and biomass by 4%–11%and 30%–111%,respectively,and the stomatal structure and carotenoids content of leaves,while decreasing the contents of chlorophyll a and b,by 21%–41%and 63%–73%,respectively.Both the MSR and MBR treatments significantly increased the photosynthetic rate and transpiration rate of rice leaves.The MAR treatment weakened chlorophyll fluorescence parameters,including the actual photosystem II(PS II)photochemical efficiency,electron transport rate,photochemical quenching,and nonphotochemical quenching by 40%,39%,43%,and 24%,respectively.Moreover,the treatments of MAR,MSR,and MBR decreased the phytohormones content and the M.oryzae disease index by 27%–62%in rice leaves.Thus,the enhanced UV-B radiation contributed to suppressing the M.oryzae infection and alleviating its damage to the photosynthesis of rice leaves.This study is valuable for the control of rice blast fungus and offers important insights into plant pathology.

Two novel gene-specific markers at the Pik locus facilitate the application of rice blast resistant alleles in breeding

Blast,a disease caused by Magnaporthe oryzae,is a major constraint for rice production worldwide.Introgression of durable blast resistance genes into high-yielding rice cultivars has been considered a priority to control the disease.The blast resistance Pik locus,located on chromosome 11,contains at least six important resistance genes,but these genes have not been widely employed in resistance breeding since existing markers hardly satisfy current breeding needs due to their limited scope of application.In this study,two PCR-based markers,Pikp-Del and Pi1-In,were developed to target the specific In Del(insertion/deletion)of the Pik-p and Pi-1 genes,respectively.The two markers precisely distinguished Pik-p,Pi-1,and the K-type alleles at the Pik locus,which is a necessary element for functional genes from rice varieties.Results also revealed that only several old varieties contain the two genes,of which nearly half carry the K-type alleles.Therefore,these identified varieties can serve as new gene sources for developing blast resistant rice.The two newly developed markers will be highly useful for the use of Pik-p,Pi-1 and other resistance genes at the Pik locus in markerassisted selection(MAS)breeding programs.

The rice miR171b–SCL6-IIs module controls blast resistance,grain yield,and flowering

Micro RNAs(mi RNAs)act as regulators of plant development and multiple stress responses.Here we demonstrate that the rice mi R171 b-SCL6-IIs module regulates the balance between blast resistance,grain yield,and flowering.mi R171 b-overexpressing rice plants(OX171 b)displayed increased rice blast resistance accompanied with enhanced defense responses and late heading,whereas blocking mi R171 b expression in rice(MIM171)led to greater susceptibility to blast disease,associated with compromised defense responses and early heading.Either overexpressing or silencing of mi R171 b significantly affected plant height and number of filled seeds per panicle(seed-setting rate),resulting in decreased grain yield.mi R171 b targets SCL6-IIa,SCL6-IIb,and SCL6-IIc,whose expression was suppressed in OX171 b but increased in MIM171.Mutants of SCL6-IIa,SCL6-IIb,and SCL6-IIc all displayed phenotypes like that of OX171 b,including markedly increased blast disease resistance,slightly decreased grain yield,and delayed flowering.Amounts of mi R171 b increased gradually in leaves during the vegetative stage but decreased gradually in panicles during the reproductive stage,whereas SCL6-IIs displayed the reverse expression pattern.Together,these results suggest that the expression of mi R171 b was time-and space-dependent during the rice growth period and regulated the balance between rice blast disease resistance,grain yield,and flowering via SCL6-IIs,and that appropriate accumulation of mi R171 b is essential for rice development.

Application Technology of Tebuconazole WG against Sheath Blight 75% Trifloxystrobin · Rice Blast and Rice

[ Objective] The paper was to confirm the best application period and frequency of 75% trifloxystrobin ·tebuconazole WG against rice blast and rice sheath blight. [ Method] Influences of different dosages and different application periods of 75% trifloxystrobin · tebuconazole WG on control effects against rice blast and rice sheath blight, as well as their effects on rice yields were studied in the paper. [Result] The control effects of three different fungicides application treatments against rice sheath blight were 80.24%, 83.0% and 67.99%, and the control effects against rice blast were 56.4%, 49.11% and 61.1%, respective- ly. Advanced application of fungicide for two times had good prevention effect against rice sheath blight ; properly delayed application of fungicides for two times was conducive to improving the control effect against rice blast, and one time application of sufficient fungicide during middle booting stage had higher control effect than application for two times. Effective panicle number per unit area, total grain number, 1 000-grain weight and moisture content of various fungicide application treat- ments did not have significant difference with those of the treatments without fungicide application, but grain number per panicle in treatments applied with fungicide was higher than the treatment without application. [ Conclusion] 75% Trifloxystrobin ·tebuconazole WG has better control effect on rice blast and sheath blight, which helps to promote the formation of rice grain and increases yield significantly. The fungicide application against rice sheath blight should be appropriately ad- vanced, and application for one time against panicle blast after middle booting stage is helpful to improve the control effect.

In-vitro vs in-vivo Inoculation: Screening for Resistance of Australian Rice Genotypes Against Blast Fungus

To assist with rapid screening for rice blast resistance as a precursor in a breeding program, the susceptibility to rice blast of 13 rice genotypes from Australia was evaluated in May to June 2013 using three distinct inoculation methods（spot, filter paper and standard methods） at seedling, vegetative and reproductive stages. The results revealed that the spot and filter paper inoculation methods were successful in discerning susceptibility to the rice blast disease（P ≤ 0.05）. Disease susceptibility declined significantly from the vegetative to reproductive stages. The standard method was conducted at three different stages for pot plants grown inside the mist house. However, low temperatures did not produce disease symptoms except in a few genotypes. Among the 13 rice genotypes screened, AAT9 expressed a highly resistant response, and AAT4, AAT6, AAT10, AAT11, AAT13, AAT17 and AAT18 expressed resistance at various stages. The results will be useful for selecting elite genotypes for disease tolerance where rice blast is prevalent. In addition, the resistant genotypes can serve as a gene pool used in breeding programmes to develop new resistant genotypes.

Weedy red rice has novel sources of resistance to biotic stress

Weedy red rice(Oryza sativa;WRR),a close relative of cultivated rice,is a highly competitive weed that commonly infests rice fields and can also naturally interbreed with rice.Useful genes for biotic stress have been maintained in WRR and can be explored for breeding.Here we describe genetic and physiological traits of WRR that can be beneficial in preventing major rice diseases.Rice blast,caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae,and sheath blight disease,caused by the necrotrophic pathogen Rhizoctonia solani,are the two most damaging biotic stresses of rice.Many major and minor resistance genes and QTL have been identified in cultivated and wild rice relatives.However,novel QTL were recently found in the two major U.S.biotypes of WRR,blackhull-awned(BH)and strawhullawnless(SH),suggesting that WRR has evolved novel genetic mechanisms to cope with these biotic stresses.Twenty-eight accessions of WRR(PI 653412–PI 653439)from the southern USA were characterized and placed in the National Small Grains Collection,and are available for identification of novel genetic factors to prevent biotic stress.

Analysis of the Resistance Gene Analogue for Rice Cultivars in Yunnan Province

Genetic diversity of commercial and local rice cultivars in Yunnan Province was studied using the resistance gene analogue (RGA) based on resistance gene conserved sequences. The RGA analysis of 137 cultivars was conducted by PCR amplification using three primers, i.e. S1/AS3, XLRR for/XLRR rev, and Pto-kinl/Pto-kin2, respectively. The results showed that both Indica and Japonica cultivars were genetically highly diverse. All cultivars were divided into 3 lineages according to the DNA band data at 96% dissimilarity, and into 20 lineages at 60% dissimilarity. The lineages were related to their genetic background and blast disease resistance with only a few exceptions. The RGA data can be useful in rice production by mixed-planting of different cultivars in the field and breeding of resistance cultivars by selecting different parental cultivars with great genetic diversity.

Identification and characterization of rice blast resistance gene Pid4 by a combination of transcriptomic profiling and genome analysis

Map-based cloning of plant disease resistance (R) genes is time-consuming. Here, we reported the isolation of blast R gene Pid4 using comparative transcriptomic profiling and genome-wide sequence analysis. Pid4 encodes a coiled-coil nucleotide-binding site leucine-rich repeat(CC-NBS-LRR) protein and is constitutively expressed at diverse developmental stages in the rice variety Digu. The Pid4 protein is localized in both the nucleus and cytoplasm. Introduction of Pid4 into susceptible rice cultivars confers race-specific resistance to leaf and neck blast. Amino acid sequence comparison and blast resistance spectrum tests showed that Pid4 is a novel R gene, different from the previously reported R genes located in the same gene cluster. A Pid4 Indel marker was developed to facilitate the identification of Pid4 in different rice varieties. We demonstrated that a plant R gene can be quickly isolated using transcriptomic profiling coupled with genome-wide sequence analysis.

The ANIP1-OsWRKY62 module regulates both basal defense and Pi9-mediated immunity against Magnaporthe oryzae in rice

During effector-triggered immunity(ETI)against the devastating rice blast pathogen Magnaporthe oryzae,Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice.Importantly,the underlying recognition mechanism(s)between Pi9 and AvrPi9 remains elusive.In this study,We identified a rice ubiquitin-like domain-containing protein(UDP),AVRPI9-INTERACTING PROTEIN 1(ANP1),which is directly targeted by AvrPi9 and also binds to Pi9 in plants.Phenotypic analysis of anip1 mu-tants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M.oryzae.ANiP1 undergoes 26S proteasome-mediated degradation,which can be blocked by both AvrPi9 and Pi9.Moreover,ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62,which also interacts with AvrPi9 and Pi9 in plants.In the absence of Pi9,ANIP1 negatively regulates OsWRKY62 abundance,which can be promoted by AvrPi9.Accordingly,knocking out of OsWRKY62 in a non-Pi9 back-ground decreased immunity against M.oryzae.However,we also observed that OsWRKY62 plays negative roles in defense against a compatible M.oryzae strain in Pi9-harboring rice.Pi9 binds to ANiP1 and OsWRKY62 to form a complex,which may help to keep Pi9 in an inactive state and weaken rice immunity.Furthermore,using competitive binding assays,we showed that AvrPi9 promotes Pi9 dissociation from ANiP1,which could be an important step toward ETI activation.Taken together,our results reveal an immune strategy whereby a UDP-WRKY module,targeted by a fungal effector,modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein.