Characterization of the leaf rust responsive ARF genes in wheat (Triticum aestivum L.).

KEY MESSAGE: Genome-wide identification, classification, functional characterization and expression analysis of Auxin Responsive Factor (ARF) gene family in wheat reveal their attributes and role during leaf rust infection. Auxins are important plant growth regulators that also impact plant-pathogen interaction. Auxin responsive factors (ARF) are plant specific transcription factors that control responses to auxins. Whole genome investigation of ARF gene family is limited in allohexaploid wheat (Triticum aestivum L.). Comprehensive study of this gene family was carried out by employing the currently available reference genome sequence of wheat. In total, 27 ARF genes were identified and located on the wheat genome as well as were positioned on wheat chromosome arms. Additionally, examination of the predicted genes unveiled a decent degree of relatedness within and among the phylogenetic clades. Leaf rust, caused by the obligate biotrophic fungal pathogen Puccinia triticina, is responsible for drastic loss of wheat crop worldwide reducing grain yield by 10-90%. Expression profiling of ARF genes in retort to leaf rust infection indicated their differential regulation during this plant-pathogen interaction. Highest expression of ARF genes were observed at 12 hpi that was maintained up to 72 hpi during incompatible interaction, whereas the high expression levels receded at 48 hpi during compatible interactions. Few of the identified ARF genes were likely to be post-transcriptionally regulated by microRNAs. Many light and stress responsive elements were detected in the promoter regions of ARF genes. Microsynteny analysis showed the conservation of ARF genes within the members of the Poaceae family. This study provides fundamental details for understanding the different types of ARF genes in wheat and there putative roles during leaf rust-wheat interaction.

Transcriptome-wide analysis of WRKY transcription factors in wheat and their leaf rust responsive expression profiling.

WRKY, a plant-specific transcription factor family, has important roles in pathogen defense, abiotic cues and phytohormone signaling, yet little is known about their roles and molecular mechanism of function in response to rust diseases in wheat. We identified 100 TaWRKY sequences using wheat Expressed Sequence Tag database of which 22 WRKY sequences were novel. Identified proteins were characterized based on their zinc finger motifs and phylogenetic analysis clustered them into six clades consisting of class IIc and class III WRKY proteins. Functional annotation revealed major functions in metabolic and cellular processes in control plants; whereas response to stimuli, signaling and defense in pathogen inoculated plants, their major molecular function being binding to DNA. Tag-based expression analysis of the identified genes revealed differential expression between mock and Puccinia triticina inoculated wheat near isogenic lines. Gene expression was also performed with six rust-related microarray experiments at Gene Expression Omnibus database. TaWRKY10, 15, 17 and 56 were common in both tag-based and microarray-based differential expression analysis and could be representing rust specific WRKY genes. The obtained results will bestow insight into the functional characterization of WRKY transcription factors responsive to leaf rust pathogenesis that can be used as candidate genes in molecular breeding programs to improve biotic stress tolerance in wheat.

Functional and DNA-protein binding studies of WRKY transcription factors and their expression analysis in response to biotic and abiotic stress in wheat (Triticum aestivum L.).

WRKY, a plant-specific transcription factor family, plays vital roles in pathogen defense, abiotic stress, and phytohormone signalling. Little is known about the roles and function of WRKY transcription factors in response to rust diseases in wheat. In the present study, three TaWRKY genes encoding complete protein sequences were cloned. They belonged to class II and III WRKY based on the number of WRKY domains and the pattern of zinc finger structures. Twenty-two DNA-protein binding docking complexes predicted stable interactions of WRKY domain with W-box. Quantitative real-time-PCR using wheat near-isogenic lines with or without Lr28 gene revealed differential up- or down-regulation in response to biotic and abiotic stress treatments which could be responsible for their functional divergence in wheat. TaWRKY62 was found to be induced upon treatment with JA, MJ, and SA and reduced after ABA treatments. Maximum induction of six out of seven genes occurred at 48 h post inoculation due to pathogen inoculation. Hence, TaWRKY (49, 50, 52, 55, 57, and 62) can be considered as potential candidate genes for further functional validation as well as for crop improvement programs for stress resistance. The results of the present study will enhance knowledge towards understanding the molecular basis of mode of action of WRKY transcription factor genes in wheat and their role during leaf rust pathogenesis in particular.

Insights of Lr28 mediated wheat leaf rust resistance: Transcriptomic approach.

Leaf rust is a fungal disease that causes severe yield losses in wheat. Resistant varieties with major and quantitative resistance genes are the most effective method to control the disease. However, the main problem is inadequate information for understanding resistance mechanism and its usefulness. This paper presents Lr28 mediated genome-wide response of known and unknown genes during wheat-Puccinia triticina interaction. In this study, we prepared Serial Analysis of Gene Expression (SAGE) libraries using seedling wheat mRNA for infected and mock conditions. The libraries were sequenced on Sequencing by Oligonucleotide Ligation and Detection (SOLiD) system generating 37-48 million reads. After mapping and gene expression analysis of ~6-12 million trimmed reads/library, we revealed five major categories comprised of Lr28 controlled transcripts in resistant (+Lr28) isoline (39), transcripts specific to susceptible (-Lr28) isoline (785), transcripts specific to hypersensitive-response (HR) (375), transcripts specific for basal-defense (153) and transcripts for establishment of pathogen (1616). We estimated the impact of specific genes and pathways through mapping on plant resistant gene database (PRGdb), reactive oxygen species (ROS) and phytohormone database. Functional annotation results revealed, receptor binding, homeostatic processes and cytoskeletal components as the major discriminating factors between susceptibility and resistance. We validated 28 key genes using qRT-PCR and found positive results. These findings were projected on hypothetical interaction model to demonstrate interaction mechanism. The study might have significant impact on future rust-resistance breeding through knowledge based smart genetic selection of quantitative resistance genes besides major effect R-gene.

Functional characterisation of a WRKY transcription factor of wheat and its expression analysis during leaf rust pathogenesis.

WRKY proteins are a large family of plant-specific transcription factors associated with regulation of biotic and abiotic stress responses, but how they respond to cereal rust pathogens has never been explored at the molecular level. Full-length cDNA of TaWRKY1B was obtained from a wheat cultivar HD2329 derivative containing leaf rust resistance gene Lr28 based on domain characteristics. The unique feature of this WRKY transcription factor gene was the close proximity of the DNA-binding domain and consensus DNA element W-Box within the open reading frame. Infection with a virulent race of leaf rust fungus resulted in 146-fold induction of the gene in resistant plants, but only 12-fold in the susceptible plants as compared with mock-inoculated controls. Docking models of 74 amino acids DNA-binding domain and 26bp W-Box element showed that the WRKY domain, located on the ß1 strand, only interacts with the W-Box at positions corresponding to W125, R126, K127 and Y128 amino acids. A truncated recombinant protein of 9.0 kD, encompassing the DNA-binding domain also showed binding specificity to the 32bp W-Box element in electrophoretic mobility shift assays. The protein-DNA ensemble was also characterised using high-resolution atomic force microscopic imaging. The results contribute to an understanding of the molecular structure and function of a previously uncharacterised WRKY transcription factor in wheat that can be manipulated to improve biotic stress tolerance.