Wheat leaf rust caused by Puccinia triticina (Pt) is one of the most common and severe diseases in the wheat-growing regions worldwide. The yield losses of wheat infected with Pt ranges from 5% − 15% and the yield of wheat infected with leaf rust at the seedling stage can be reduced by 50% or even more [1]. Considering the impacts of global climate change, temperature and humidity conditions may be more suitable for the proliferation and epidemic of Pt in the future which will further deleteriously impact wheat yields. Control of leaf rust mainly relies on the fungicide application and deployment of cultivars carrying resistance genes. Genetic resistance is the most effective, environmentally safe, and economically feasible approach to reduce the damage caused by Pt. However, monoculture of select resistant varieties leads to the host selection pressures that drive Pt evolution and promote the continuous emergence of new toxic races, which often leads to the decline of wheat resistant varieties after several years of planting.
To date, over 80 genes conferring leaf rust resistance derived from Triticum aestivum wheat cultivars, wild wheat, grass species, and durum wheat have been identified and designated as Lr1 to Lr79 [2]. Among them, only Lr9, Lr19, Lr24, Lr34, Lr37, Lr38, Lr46, Lr47, Lr51, Lr53, and Lr68 confer effective resistance to leaf rust currently. Lr19 is derived from the grass Agropyron elongatum and was transferred to the long arm of wheat chromosome 7D [3]. The wheat cultivar carrying Lr19 is still effective against all Pt races in Asia, Australia, Canada, and Europe, and resistance is expressed during the whole growth period [4, 5]. Therefore, Lr19 holds the potential to be deployed in combination with other Lr genes in the field to confer durable resistance against leaf rust worldwide [4, 6]. It has been reported that Lr19 is associated with increased grain yield, which promotes it as an important gene for wheat breeding against Pt-mediate yield losses [7]. To prevent Lr19 from being out-competed by newly emergent virulent races, cloning avirulent gene AvrLr19 that can be recognized by wheat leaf rust resistance gene Lr19, determining the molecular mechanism of AvrLr19 and Lr19 interactions, and monitoring the natural Pt population changes in response to AvrLr19-Lr19 resistance are key to enable the long-term deployment of Lr19 in leaf rust-resistant wheat varieties. At present, there is no virulent strain of Lr19 in the field, which not only benefits the use of Lr19 but also restricts the development of AvrLr19.
Mutation is the most important avenue for creating new rust races and genotypes. Ethyl methanesulfonate (EMS) is an alkylating chemical mutagen that generates single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels), resulting in amino acid sequence variation, and finally leads to phenotype changes that can be selected upon. Mutagenesis integrated with genomic sequencing is an efficient way to study the relationships between phenotypic traits and associated genotypes, leading to the identification of fungal effectors or Avr genes. For example, Salcedo et al. obtained stripe rust mutants through EMS-induced mutation and performed genome sequencing to obtain AvrSr35 candidate genes, and then verified the candidate genes through co-expression of AvrSr35 and its corresponding resistance gene in tobacco and wheat to trigger cell death [8]. Li et al. screened 30 mutant variants from the least virulent isolate generated by EMS mutagenesis and candidates Avr genes were determined by sequencing [9]. Transcriptomics has proven to be an instrumental molecular tool to help identify virulence effectors and Avr genes [10–12]. As the host and pathogen interact in a battle for supremacy, the underlying transcriptional regulation of gene expression in the plant and pathogen provides clues to their defense and virulence mechanisms, respectively [13].
The gene to gene hypothesis proposed by Flor indicates that only the host with an “R gene” is resistant to the homologous Avr gene in pathogens [14]. Jone proposed the famous “ZigZag” model in 2006 to analyze the molecular mechanism of interaction between plants and their pathogens [15]. In order to inhibit plant defense responses, pathogens secrete a series of effector proteins through the haustorium to interfere with or ablate the plant defense response, so as to meet their own growth needs. In recent years, with the continuous improvement of sequencing technology and the reduction of sequencing costs, and the development and application of prediction software such as SigalP [16], TargetP [17], TMHMM [18], PredGPI [19] and Pfam [20] have improved the screening efficiency of candidate Avr genes of rust. To date, a handful of Avr genes have been identified in rust pathogens, including AvrL567, AvrP123, AvrP4, AvrM, AvrL2, and AvrM14 from the flax rust pathogen [21], as well as RTP1 in bean rust [22], and PGTAUSPE10–1 from wheat stem rust [23]. At the end of 2017, two articles reported the successful cloning of the Avr genes AvrSr35 [8] and AvrSr50 [24] of wheat stem rust, and preliminarily revealed their interaction with corresponding resistance genes. Some effector candidates for Lr26, Lr9, and Lr24 materials were obtained from Pt [25] and 20 effector candidates of Lr20 [26], but their biological functions have not been determined, so, no known Avr genes have been identified in Pt so far.
In this study, we aim to use an EMS mutagenized Pt race PHNT to obtain Lr19-virulent mutants, and identify differentially expressed genes (DEGs) associated with the Pt infection. Our results provide resources for identification of AvrLr19 candidates and pathogenicity-related genes, and lay a foundation for revealing the pathogenic mechanism of Pt.