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Unraveling Effects of miRNAs Associated with APR Leaf Rust Resistance Genes in Hybrid Forms of Common Wheat (Triticum aestivum L.)
The fungus Puccinia triticina Eriks (Pt) is the cause of leaf rust, one of the most damaging diseases, which significantly reduces common wheat yields. In Pt-resistant adult plants, an APR-type resistance is observed, which protects the plant against multiple pathogen races and is distinguished by its persistence under production conditions. With a more complete understanding of the molecular mechanisms underlying the function of APR genes, it will be possible to develop new strategies for resistance breeding in wheat. Currently, mainly APR genes, such as Lr34, Lr46, and Lr67, are principally involved in resistance breeding as they confer durable resistance to multiple fungal races occurring under different climatic and environmental conditions. However, the mechanisms underlying the defence against pathogens mediated by APR genes remain largely unknown. Our research aimed to shed light on the molecular mechanisms related to resistance genes and miRNAs expression, underlying APR resistance to leaf rust caused by Pt. Furthermore, the present study aimed to identify and functionally characterize the investigated miRNAs and their target genes in wheat in response to leaf rust inoculation. The plant material included hybrid forms of wheat from the F2 and BC1F1 generations, obtained by crossing the resistance cultivar Glenlea (CItr 17272) with agriculturally important Polish wheat cultivars. Biotic stress was induced in adult plants via inoculation with Pt fungal spores under controlled conditions. The RT-qPCR method was used to analyze the expression profiles of selected APR genes at five time points (0, 6, 12, 24, and 48 hpi). The results presented here demonstrate the differential expression of APR genes and miRNAs at stages of leaf rust development at selected timepoints after inoculation. We analyzed the expression of three leaf rust resistance genes, using different genetic backgrounds in F2 and BC1F1 segregation materials, in leaf tissues after Pt infection. Our goal was to investigate potential differences resulting from the genetic background found in different generations of hybrid forms of the same parental forms. Gene ontology analysis predicted 190 target genes for tae-miR5384-3p and 167 target genes for tae-miR9653b. Our findings revealed distinct expression profiles for genes, with the highest expression levels observed mainly at 6, 24, and 48 hpi. The candidate gene Lr46-Glu2 displayed an upregulation, suggesting its potential involvement in the immune response against Pt infection.
Diversity of Expression Patterns of Lr34, Lr67, and Candidate Genes towards Lr46 with Analysis of Associated miRNAs in Common Wheat Hybrids in Response to Puccinia triticina Fungus
Leaf rust caused by Puccinia triticina (Pt) is one of the most dangerous diseases causing significant losses in common wheat crops. In adult plants resistant to rust, a horizontal adult plant resistance (APR) type is observed, which protects the plant against multiple pathogen races and is distinguished by greater persistence under production conditions. Crucial pleiotropic slow-rust genes such as Lr34, Lr46, Lr67, and Lr68, in combination with other genes of lesser influence, continue to increase durable resistance to rust diseases. Based on our previous results, we selected four candidate genes for Lr46 out of ten candidates and analysed them for expression before and after inoculation by P. triticina. As part of our study, we also investigated the expression patterns of miRNA molecules complementary to Lr34 and the candidate genes. The aim of the study was to analyse the expression profiles of candidate genes for the Lr46 gene and the Lr34 and Lr67 genes responsible for the differential leaf-rust resistance of hybrid forms of the F1 generation resulting from crosses between the Glenlea cultivar and cultivars from Polish breeding companies. In addition, the expression of five miRNAs (tae-miR9653b, tae-miR5384-3p, tae-miR9780, tae-miR9775 and tae-miR164), complementary to Lr34, and selected candidate genes were analysed using stem-loop RT-PCR and ddPCR. Biotic stress was induced in adult plants by inoculation with Pt fungal spores, under controlled conditions. Plant material was collected before and 6, 12, 24, and 48 h after inoculation (hpi). Differences in expression patterns of Lr34, Lr67, and candidate genes (for Lr46) were analysed by qRT-PCR and showed that gene expression changed at the analysed time points. Identification of molecular markers coupled to the Lr genes studied was also carried out to confirm the presence of these genes in wheat hybrids. qRT-PCR was used to examine the expression levels of the resistance genes. The highest expression of Lr46/Yr29 genes (Lr46-Glu2, Lr46-RLK1, Lr46-RLK2, and Lr46-RLK3) occurred at 12 and 24 hpi, and such expression profiles were obtained for only one candidate gene among the four genes analysed (Lr46-Glu2), indicating that it may be involved in resistance mechanisms of response to Pt infection.
Expression Profiling of the Slow Rusting Resistance Genes Lr34/Yr18 and Lr67/Yr46 in Common Wheat (Triticum aestivum L.) and Associated miRNAs Patterns
The main efforts in common wheat (Triticum aestivum L.) breeding focus on yield, grain quality, and resistance to biotic and abiotic stresses. One of the major threats affecting global wheat cultivation and causing significant crop production losses are rust diseases, including leaf rust caused by a biotrophic fungus Puccinia triticina Eriks. Genetically determined resistance to leaf rust has been characterized in young plants (seedling resistance) as well as in plants at the adult plant stage. At the seedling stage, resistance is controlled vertically by major R genes, conferring a race-specific response that is highly effective but usually short-lived due to the rapid evolution of potentially virulent fungi. In mature plants, horizontal adult plant resistance (APR) was described, which provides long-term protection against multiple races of pathogens. A better understanding of molecular mechanisms underlying the function of APR genes would enable the development of new strategies for resistance breeding in wheat. Therefore, in the present study we focused on early transcriptomic responses of two major wheat APR genes, Lr34 and Lr67, and three complementary miRNAs, tae-miR9653b, tae-miR9773 and tae-miR9677b, to inoculation with P. triticina. Plant material consisted of five wheat reference varieties, Artigas, NP846, Glenlea, Lerma Rojo and TX89D6435, containing the Lr34/Yr18 and Lr67/Yr46 resistance genes. Biotic stress was induced by inoculation with fungal spores under controlled conditions in a phytotron. Plant material consisted of leaf tissue sampled before inoculation as well as 6, 12, 24 and 48 h postinoculation (hpi). The APR gene expression was quantified using real-time PCR with two reference genes, whereas miRNA was quantified using droplet digital PCR. This paper describes the resistance response of APR genes to inoculation with races of leaf rust-causing fungi that occur in central Europe. The study revealed high variability of expression profiles between varieties and time-points, with the prevalence of downregulation for APR genes and upregulation for miRNAs during the development of an early defense response. Nevertheless, despite the downregulation initially observed, the expression of Lr34 and Lr67 genes in studied cultivars was significantly higher than in a control line carrying wild (susceptible) alleles.