Weijian Zhuang

Functional genomics to study stress responses in crop legumes: progress and prospects

Legumes are important food crops worldwide, contributing to more than 33% of human dietary protein. The production of crop legumes is frequently impacted by abiotic and biotic stresses. It is therefore important to identify genes conferring resistance to biotic stresses and tolerance to abiotic stresses that can be used to both understand molecular mechanisms of plant response to the environment and to accelerate crop improvement. Recent advances in genomics offer a range of approaches such as the sequencing of genomes and transcriptomes, gene expression microarray as well as RNA-seq based gene expression profiling, and map-based cloning for the identification and isolation of biotic and abiotic stress-responsive genes in several crop legumes. These candidate stress associated genes should provide insights into the molecular mechanisms of stress tolerance and ultimately help to develop legume varieties with improved stress tolerance and productivity under adverse conditions. This review provides an overview on recent advances in the functional genomics of crop legumes that includes the discovery as well as validation of candidate genes.

Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco

Summary Bacterial wilt caused by Ralstonia solanacearum is a ruinous soilborne disease affecting more than 450 plant species. Efficient control methods for this disease remain unavailable to date. This study characterized a novel nucleotide‐binding site‐leucine‐rich repeat resistance gene AhRRS5 from peanut, which was up‐regulated in both resistant and susceptible peanut cultivars in response to R. solanacearum. The product of AhRRS5 was localized in the nucleus. Furthermore, treatment with phytohormones such as salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (ET) increased the transcript level of AhRRS5 with diverse responses between resistant and susceptible peanuts. Abiotic stresses such as drought and cold conditions also changed AhRRS5 expression. Moreover, transient overexpression induced hypersensitive response in Nicotiana benthamiana. Overexpression of AhRRS5 significantly enhanced the resistance of heterogeneous tobacco to R. solanacearum, with diverse resistance levels in different transgenic lines. Several defence‐responsive marker genes in hypersensitive response, including SA, JA and ET signals, were considerably up‐regulated in the transgenic lines as compared with the wild type inoculated with R. solanacearum. Nonexpressor of pathogenesis‐related gene 1 (NPR1) and non‐race‐specific disease resistance 1 were also up‐regulated in response to the pathogen. These results indicate that AhRRS5 participates in the defence response to R. solanacearum through the crosstalk of multiple signalling pathways and the involvement of NPR1 and R gene signals for its resistance. This study may guide the resistance enhancement of peanut and other economic crops to bacterial wilt disease.

Oxidative stress and carbon metabolism influence Aspergillus flavus transcriptome composition and secondary metabolite production

Contamination of crops with aflatoxin is a serious global threat to food safety. Aflatoxin production by Aspergillus flavus is exacerbated by drought stress in the field and by oxidative stress in vitro. We examined transcriptomes of three toxigenic and three atoxigenic isolates of A. flavus in aflatoxin conducive and non-conducive media with varying levels of H2O2 to investigate the relationship of secondary metabolite production, carbon source, and oxidative stress. We found that toxigenic and atoxigenic isolates employ distinct mechanisms to remediate oxidative damage, and that carbon source affected the isolates’ expression profiles. Iron metabolism, monooxygenases, and secondary metabolism appeared to participate in isolate oxidative responses. The results suggest that aflatoxin and aflatrem biosynthesis may remediate oxidative stress by consuming excess oxygen and that kojic acid production may limit iron-mediated, non-enzymatic generation of reactive oxygen species. Together, secondary metabolite production may enhance A. flavus stress tolerance, and may be reduced by enhancing host plant tissue antioxidant capacity though genetic improvement by breeding selection.

Peanut Aflatoxin and Genomics Research in China: Progress and Perspectives

Peanut is an important oil and food crop in China with a unique role in agricultural development and food security. Aflatoxin contamination in peanut, normally more serious in southern parts of the country, is a crucial factor affecting sustainable development of the peanut industry. Extensive efforts have been made at several institutions in China for aflatoxin management and related genomics research. Several local peanut germplasm lines have been identified as resistant to seed infection by Aspergillus flavus or aflatoxin production. Two AFLP markers have been identified that are linked with resistance to seed invasion and one was converted into a SCAR marker for more efficient breeding application. Several new peanut cultivars with improved productivity and possessing resistance to aflatoxin contamination are extensively used in production. Integrated management approaches have been recommended to farmers based on agro-ecological characteristics in different regions. More recently, molecular techniques have been extensively used in genetic diversity assessment, investigation of genetic relationships among different germplasm groups, marker development, identification of interspecific genome introgression, gene cloning and function analysis, and genetic transformation of important traits concerning productivity, quality and food safety of peanut. Special emphasis has been placed on resistance to aflatoxin, bacterial wilt, foliar diseases and fatty acid desaturase. Perspectives of peanut genetic improvement and further research priorities are also discussed.

Mapping Quantitative Trait Loci of Resistance to Tomato Spotted Wilt Virus and Leaf Spots in a Recombinant Inbred Line Population of Peanut (Arachis hypogaea L.) from SunOleic 97R and NC94022

Peanut is vulnerable to a range of diseases, such as Tomato spotted wilt virus (TSWV) and leaf spots which will cause significant yield loss. The most sustainable, economical and eco-friendly solution for managing peanut diseases is development of improved cultivars with high level of resistance. We developed a recombinant inbred line population from the cross between SunOleic 97R and NC94022, named as the S-population. An improved genetic linkage map was developed for the S-population with 248 marker loci and a marker density of 5.7 cM/loci. This genetic map was also compared with the physical map of diploid progenitors of tetraploid peanut, resulting in an overall co-linearity of about 60% with the average co-linearity of 68% for the A sub-genome and 47% for the B sub-genome. The analysis using the improved genetic map and multi-season (2010–2013) phenotypic data resulted in the identification of 48 quantitative trait loci (QTLs) with phenotypic variance explained (PVE) from 3.88 to 29.14%. Of the 48 QTLs, six QTLs were identified for resistance to TSWV, 22 QTLs for early leaf spot (ELS) and 20 QTLs for late leaf spot (LLS), which included four, six, and six major QTLs (PVE larger than 10%) for each disease, respectively. A total of six major genomic regions (MGR) were found to have QTLs controlling more than one disease resistance. The identified QTLs and resistance gene-rich MGRs will facilitate further discovery of resistance genes and development of molecular markers for these important diseases.

Analysis of genetic diversity and population structure of peanut cultivars and breeding lines from China, India and the US using simple sequence repeat markers

Cultivated peanut is grown worldwide as rich-source of oil and protein. A broad genetic base is needed for cultivar improvement. The objectives of this study were to develop highly informative simple sequence repeat (SSR) markers and to assess the genetic diversity and population structure of peanut cultivars and breeding lines from different breeding programs in China, India and the US. A total of 111 SSR markers were selected for this study, resulting in a total of 472 alleles. The mean values of gene diversity and polymorphic information content (PIC) were 0.480 and 0.429, respectively. Country-wise analysis revealed that alleles per locus in three countries were similar. The mean gene diversity in the US, China and India was 0.363, 0.489 and 0.47 with an average PIC of 0.323, 0.43 and 0.412, respectively. Genetic analysis using the STRUCTURE divided these peanut lines into two populations (P1, P2), which was consistent with the dendrogram based on genetic distance (G1, G2) and the clustering of principal component analysis. The groupings were related to peanut market types and the geographic origin with a few admixtures. The results could be used by breeding programs to assess the genetic diversity of breeding materials to broaden the genetic base and for molecular genetics studies.

Proteome analysis of Aspergillus flavus isolate-specific responses to oxidative stress in relationship to aflatoxin production capability

Aspergillus flavus is an opportunistic pathogen of plants such as maize and peanut under conducive conditions such as drought stress resulting in significant aflatoxin production. Drought-associated oxidative stress also exacerbates aflatoxin production by A. flavus. The objectives of this study were to use proteomics to provide insights into the pathogen responses to H2O2-derived oxidative stress, and to identify potential biomarkers and targets for host resistance breeding. Three isolates, AF13, NRRL3357, and K54A with high, moderate, and no aflatoxin production, were cultured in medium supplemented with varying levels of H2O2, and examined using an iTRAQ (Isobaric Tags for Relative and Absolute Quantification) approach. Overall, 1,173 proteins were identified and 220 were differentially expressed (DEPs). Observed DEPs encompassed metabolic pathways including antioxidants, carbohydrates, pathogenicity, and secondary metabolism. Increased lytic enzyme, secondary metabolite, and developmental pathway expression in AF13 was correlated with oxidative stress tolerance, likely assisting in plant infection and microbial competition. Elevated expression of energy and cellular component production in NRRL3357 and K54A implies a focus on oxidative damage remediation. These trends explain isolate-to-isolate variation in oxidative stress tolerance and provide insights into mechanisms relevant to host plant interactions under drought stress allowing for more targeted efforts in host resistance research.

AhRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase of peanut, confers increased resistance to bacterial wilt in tobacco

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease that infects hundreds of plant species. Host factors involved in disease resistance and pathogenesis remain poorly characterized. An up regulated and leucine-rich repeat receptor-like kinase characterized as CLAVATA1 and named AhRLK1 was obtained by microarray analysis in response to R. solanacearum in peanut. AhRLK1 contained presumably, a signal peptide, ten leucine-rich repeat (LRR) domains and conserved motifs of intracellular kinases. For subcellular localization, the AhRLK1 protein was visualized only in the plasma membrane. After inoculation with R. solanacearum, AhRLK1 was constantly up regulated in the susceptible variety Xinhuixiaoli but showed little changed in the resistant cultivar Yueyou92. Different hormones, including salicylic acid, abscisic acid, methyl jasmonate, and ethephon, induced expression, but expression was completely down regulated under cold and drought treatments. Transient overexpression provoked a hypersensitive response (HR) in Nicotiana benthamiana following agro-infiltration. Furthermore, in transgenic tobacco with overexpression of the gene, the resistance to R. solanacearum increased significantly. By contrast, most representative defense-responsive genes in HR, SA, JA and ET signal pathways such as NtHIN1, NtPR2, NtLOX1, and NtACS6, among others, were considerably up regulated in the AhRLK1 transgenic lines. Additionally, the EDS1 and PAD4 in the R gene signal were also up regulated in transgenic plants, but the NDR1 and NPR1 genes were down regulated. Accordingly, we suggest that AhRLK1 increases the defense response to R. solanacearum via HR and hormone defense signalling, associated with the EDS1 pathway of R gene signalling. The results provide new understanding of CLV1 function and will contribute to genetic enhancement of peanut.