Silencing GmWRKY33B genes leads to reduced disease resistance in soybean / 生物工程学报

The WRKYs are a group of plant-specific transcription factors that play important roles in defense responses. In this study, we silenced 2 GmWRKY33B homologous genes using a bean pod mosaic virus (BPMV) vector carrying a single fragment from the conserved region of the GmWRKY33B genes. Silencing GmWRKY33B did not result in morphological changes. However, significantly reduced resistances to Pseudomonas syringae pv. glycinea (Psg) and soybean mosaic virus (SMV) were observed in the GmWRKY33B-silenced plants, indicating a positive role of the GmWRKY33B genes in disease resistance. Kinase assay showed that silencing the GmWRKY33B genes significantly reduced the activation of GmMPK6, but not GmMPK3, in response to flg22 treatment. Reverse transcriptase PCR (RT-PCR) analysis of the genes encoding prenyltransferases (PTs), which are the key enzymes in the biosynthesis of glyceollin, showed that the Psg-induced expression of these genes was significantly reduced in the GmWRKY33B-silenced plants compared with the BPMV-0 empty vector plants, which correlated with the presence of the W-boxes in the promoter regions of these genes. Taken together, our results suggest that GmWRKY33Bs are involved in soybean immunity through regulating the activation of the kinase activity of GmMPK6 as well as through regulating the expression of the key genes encoding the biosynthesis of glyceollins.

Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach / Introdução do gene resistente à ferragem da folha bacteriana (BLB), xa7 na variedade MARDI elite, MR219 por abordagem de backcrossing assistida por marcador (MABC)

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

Characterization of wheat-Thinopyrum bessarabicum genetic stock for stripe rust and Karnal bunt resistance / Caracterização do estoque genético de trigo-Thinopyrum bessarabicum para resistência de ferrugem e Karnal bunt

Abstract Utilization of modern breeding techniques for developing high yielding and uniform plant types ultimately narrowing the genetic makeup of most crops. Narrowed genetic makeup of these crops has made them vulnerable towards disease and insect epidemics. For sustainable crop production, genetic variability of these crops must be broadened against various biotic and abiotic stresses. One of the ways to widen genetic configuration of these crops is to identify novel additional sources of durable resistance. In this regard crops wild relatives are providing valuable sources of allelic diversity towards various biotic, abiotic stress tolerance and quality components. For incorporating novel variability from wild relative's wide hybridization technique has become a promising breeding method. For this purpose, wheat-Th. bessarabicum amphiploid, addition and translocation lines have been screened in field and screen house conditions to get novel sources of yellow rust and Karnal bunt resistant. Stripe rust screening under field conditions has revealed addition lines 4JJ and 6JJ as resistant to moderately resistant while addition lines 3JJ, 5JJ, 7JJ and translocation lines Tr-3, Tr-6 as moderately resistant wheat-Thinopyrum-bessarabicum genetic stock. Karnal bunt screening depicted addition lines 5JJ and 4JJ as highly resistant genetic stock. These genetic stocks may be used to introgression novel stripe rust and Karnal bunt resistance from the tertiary gene pool into susceptible wheat backgrounds.

Resumo A utilização de técnicas modernas de melhoramento para o desenvolvimento de tipos de plantas uniformes e de alto rendimento, em última análise, estreitando a composição genética da maioria das culturas. A composição genética restrita dessas plantações tornou-as vulneráveis a doenças e epidemias de insetos. Para uma produção agrícola sustentável, a variabilidade genética dessas culturas deve ser ampliada contra vários estresses bióticos e abióticos. Uma das maneiras de ampliar a configuração genética dessas culturas é identificar novas fontes adicionais de resistência durável. A esse respeito, os parentes selvagens das culturas estão fornecendo fontes valiosas de diversidade alélica para vários componentes de qualidade e tolerância ao estresse abiótico e biótico. Para incorporar a nova variabilidade da ampla técnica de hibridização de parente selvagem tornou-se um método de reprodução promissor. Para esse efeito, trigo-Th. As linhas anfiploides, de adição e translocação de bessarabicum foram selecionadas em condições de campo e de casa de tela para obter novas fontes de ferrugem amarela e resistência ao bunt de Karnal. A triagem de ferrugem em faixas em condições de campo revelou as linhas de adição 4JJ e 6JJ como resistentes a moderadamente resistentes, enquanto as linhas de adição 3JJ, 5JJ, 7JJ e as linhas de translocação Tr-3, Tr-6 como estoque genético de trigo-Thinopyrum bessarabicum moderadamente resistente. A triagem Karnal bunt descreveu as linhas de adição 5JJ e 4JJ como estoque genético altamente resistente. Esses estoques genéticos podem ser usados para introgressão da nova ferrugem e resistência ao bunt de Karnal do pool genético terciário em origens de trigo suscetíveis.

Fructosan form Paenibacillus kribbensis PS04 enhance disease resistance against Rhizoctonia solani and tobacco mosaic virus

BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

Identificación de marcadores moleculares (SNP) y genes candidatos asociados a la tolerancia genética al complejo mancha de asfalto en variedades nativas de maíz (Zea mays L) / Identification of molecular markers (SNP) and candidate genes associated to genetic tolerance to tar spot complex disease in maize (Zeamays L) landraces

El complejo mancha de asfalto (CMA) en maíz (ZeamaysL.), causado por los hongos Phyllachora maydis Maubl. Y Monographella maydis Müller & Samuels, es una enfermedad de importancia económica en Guatemala, que ha causado pérdida en el rendimiento entre 30 a 50%, inclusive del 100% si las condiciones son favorables. El objetivo de esta investigación fue identificar marcadores de un solo nucleótido o SNP (Single Nucleotide Polymorphism, por sus siglas en inglés) y genes candidatos asociados a la tolerancia genética al CMA. Para ello se analizaron 463 poblaciones nativas y 329,692 SNP, y se compararon dos modelos genómicos, single markery BayesB, para la identificación de regiones asociadas a la tolerancia genética al CMA. Se identificaron 40 marcadores SNP asociados significativamente a la tolerancia genética al CMA con ambos modelos. La proporción de variación fenotípica total explicada (PVE) por los 40 SNPs fue de 56%, atribuida a efectos genéticos aditivos. Múltiples genes de resistencia a enfermedades fueron identificados en las regiones señaladas por los marcadores SNP. Sus funciones principales son receptores y transductores de señal, factores de transcripción que regulan positivamente la expresión de genes de tolerancia y genes de la familia kinasa, por lo que potencialmente están involucrados en el mecanismo de defensa al CMA.

The tar spot complex (TSC) diseasein maize (ZeamaysL.), caused by the fungi Phyllachora maydis Maubl. And Monographella maydis Müller & Samuels, is an economic important disease in Guatemala, producing yield losses between 30 to 50%, inclusive of 100% if the conditionsare favorable. The objective of this researchwasto identify single nucleoti depolymorphism markers (SNP) and candidate genes associated withgenetictoleranceto TSC. Asetof 463 native populations and 329,692 SNP were analyzed with two genomic models, single marker and BayesB, for the identification of regions associated with genetic tolerance to TSC. Forty SNP markers were significantly associated with the genetic tolerance to TSC with both models. The proportion of total phenotypic variation explained (PVE) by the 40 SNPs was 56%, attributed to additive genetice ffects. Multiple candidate genes for disease resistance were identified in the región sindicated by the SNP markers. Their main functionsare signal transducersand receptors, transcription factors that positively regulatethe expression of tolerance genes and family kinase genes, there fore, they are potentially involved in the defense mechanism to TSC.

Transcriptome and proteome analyses of resistant preharvest peanut seed coat in response to Aspergillus flavus infection

BACKGROUND: The infection of peanut (Arachis hypogaea L.) seed coat by the pathogenic fungus Aspergillus flavus has highly negative economic and health impacts. However, the molecular mechanism underlying such defense response remains poorly understood. This study aims to address this issue by profiling the transcriptomic and proteomic changes that occur during the infection of the resistant peanut cultivar J11 by A. flavus. RESULTS: Transcriptomic study led to the detection of 13,539 genes, among which 663 exhibited differential expression. Further functional analysis found the differentially expressed genes to encode a wide range of pathogenesis- and/or defense-related proteins such as transcription factors, pathogenesis-related proteins, and chitinases. Changes in the expression patterns of these genes might contribute to peanut resistance to A. flavus. On the other hand, the proteomic profiling showed that 314 of the 1382 detected protein candidates were aberrantly expressed as a result of A. flavus invasion. However, the correlation between the transcriptomic and proteomic data was poor. We further demonstrated by in vitro fungistasis tests that hevamine-A, which was enriched at both transcript and protein levels, could directly inhibit the growth of A. flavus. Conclusions: The results demonstrate the power of complementary transcriptomic and proteomic analyses in the study of pathogen defense and resistance in plants and the chitinase could play an important role in the defense response of peanut to A. flavus. The current study also constitutes the first step toward building an integrated omics data platform for the development of Aspergillus-resistant peanut cultivars

Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.

Identification of groundnut (Arachis hypogaea) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm

Background This study aimed to identify and select informative Simple Sequence Repeat (SSR) markers that may be linked to resistance to important groundnut diseases such as Early Leaf Spot, Groundnut Rosette Disease, rust and aflatoxin contamination. To this end, 799 markers were screened across 16 farmer preferred and other cultivated African groundnut varieties that are routinely used in groundnut improvement, some with known resistance traits. Results The SSR markers amplified 817 loci and were graded on a scale of 1 to 4 according to successful amplification and ease of scoring of amplified alleles. Of these, 376 markers exhibited Polymorphic Information Content (PIC) values ranging from 0.06 to 0.86, with 1476 alleles detected at an average of 3.7 alleles per locus. The remaining 423 markers were either monomorphic or did not work well. The best performing polymorphic markers were subsequently used to construct a dissimilarity matrix that indicated the relatedness of the varieties in order to aid selection of appropriately diverse parents for groundnut improvement. The closest related varieties were MGV5 and ICGV-SM 90704 and most distant were Chalimbana and 47-10. The mean dissimilarity value was 0.51, ranging from 0.34 to 0.66. Discussion Of the 376 informative markers identified in this study, 139 (37%) have previously been mapped to the Arachis genome and can now be employed in Quantitative Trait Loci (QTL) mapping and the additional 237 markers identified can be used to improve the efficiency of introgression of resistance to multiple important biotic constraints into farmer-preferred varieties of Sub-Saharan Africa.

Analysis on the arcelin expression in bruchid pest resistant wild pulses using real time RT-qPCR.

Arcelin, the antimetabolic protein from wild pulses is a known natural insecticidal molecule. Wild pulses with high arcelin content could serve as potential source to increase the levels of insect resistance in cultivated pulse crops. In this study, arcelin (Arl) gene expression was screened in seven stored product insect pest resistant wild pulse varieties using real time RT-qPCR. Arcelin gene specific real time PCR primers were synthesized from arcelin mRNA sequence of the wild pulse variety, Lablab purpureus. The results revealed different levels of arcelin gene expression in the tested varieties. Canavalia virosa registered significantly high content indicating its suitability for utilization of arcelin gene in developing stored product insect pest resistance with other cultivated pulses.

Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)

Background Head smut of maize, which is caused by Sporisorium reilianum f. sp. zeae (Kühn), is a serious disease in maize. In order to reveal the molecular mechanism of the resistance to head smut in maize, a microarray containing ~ 14,850 probes was used to monitor the gene expression profiles between a disease resistant near isogenic line (NIL) and a highly susceptible inbred line after S. reilianum was injected with an artificial inoculation method. Results Levels of expression for 3,532 genes accounting for 23.8% of the total probes changed after inoculation. Gene Ontology analysis revealed that the differentially expressed genes participated in physiological and biochemical pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that plant-pathogen interaction, natural killer cell mediated cytotoxicity and benzoxazinoid biosynthesis pathways play important roles in resistance to head smut. Three head smut resistance-related candidate genes, CLAVATA1, bassinosteroid insensitive 1-associated receptor kinase 1 and LOC100217307 with leucine-rich repeat (LRR) conserved domains were identified, each of which is in maize mapping bin 2.09, a region previously shown to include a major QTL for head smut resistance. Furthermore, LOC100217307 was validated by quantitative real-time (qRT)-PCR inferring that this gene may be involved in the resistance to head smut of maize. Conclusions This study provided valuable information for cloning, functional analysis and marker assisted breeding of head smut resistance genes.

Phenylpropanoid enzymes, phenolic polymers and metabolites as chemical defenses to infection of Pratylenchus coffeae in roots of resistant and susceptible bananas (Musa spp.).

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.

Data mining and influential analysis of gene expression data for plant resistance gene identification in tomato (Solanum lycopersicum)

Background Molecular mechanisms of plant-pathogen interactions have been studied thoroughly but much about them is still unknown. A better understanding of these mechanisms and the detection of new resistance genes can improve crop production and food supply. Extracting this knowledge from available genomic data is a challenging task. Results Here, we evaluate the usefulness of clustering, data-mining and regression to identify potential new resistance genes. Three types of analyses were conducted separately over two conditions, tomatoes inoculated with Phytophthora infestans and not inoculated tomatoes. Predictions for 10 new resistance genes obtained by all applied methods were selected as being the most reliable and are therefore reported as potential resistance genes. Conclusion Application of different statistical analyses to detect potential resistance genes reliably has shown to conduct interesting results that improve knowledge on molecular mechanisms of plant resistance to pathogens.

Resistance of citrus genotypes to Phyllocnitis citrella Stainton (Lepidoptera: Gracillariidae)

The development and reproduction of the citrus leafminer (CLM), Phyllocnistis citrella Stainton, were evaluated in six citrus genotypes in order to identify genotypes with resistance traits that could be applied in a program for the development of citrus varieties resistant to the citrus leafminer. Tests were conducted under controlled laboratory conditions (25 ± 1ºC, 70 ± 10 percent RH, and 14h photophase). Seedlings of each genotype tested were infested with eggs obtained from a stock colony of CLM maintained on 'Cravo' lemon (Citrus limonia L. Osbeck), and the duration and survival of the eggs, larval and pupal stages, pupal size and weight, fecundity and longevity of adults, and sex ratio were evaluated. No influence was observed on the duration and survival of eggs, larvae and pupae of P. citrella. However, pupae obtained in the hybrid C x R4 were significantly smaller and lighter than pupae from the remaining treatments. Adult females from the hybrids C x R4 and C x R315 were the least fecund. However, the lowest value for the corrected reproductive potential (CRP) was recorded in the hybrid C x R315, suggesting that this genotype is the least favorable for the development and reproduction of CLM. On the other hand, the highest CRP value obtained in the 'Rugoso' lemon confirms the susceptibility of this genotype, indicating it as the most suitable for CLM.