Molecular Characterization of Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Collections from Nine Countries.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. To understand the worldwide distribution of its molecular groups, as well as the diversity, differentiation, and migration of the Pst populations, 567 isolates collected from nine countries (China, Pakistan, Italy, Egypt, Ethiopia, Canada, Mexico, Ecuador, and the U.S.) in 2010-2018 were genotyped using 14 codominant simple sequence repeat markers. A total of 433, including 333 new multi-locus genotypes (MLGs), were identified, which were clustered into ten molecular groups (MGs). The MGs and country-wise populations differed in genetic diversity, heterozygosity, and correlation coefficient between the marker and virulence data. Many isolates from different countries, especially the isolates from Mexico, Ecuador, and the U.S., were found to be identical or closely related MLGs, and some of the MGs were present in all countries, indicating Pst migrations among different countries. The analysis of molecular variance revealed 78% variation among isolates, 12% variation among countries, and 10% variation within countries. Only low levels of differentiation were found by the pairwise comparisons of country populations. Of the 10 MGs, 5 were found to be involved in sexual and/or somatic recombination. Identical and closely related MLGs identified from different countries indicated international migrations. The study provides information on the distributions of various Pst genetic groups in different countries and evidence for the global migrations, which should be useful in understanding the pathogen evolution and in stressing the need for continual monitoring of the disease and pathogen populations at the global scale.

In-depth secretome analysis of Puccinia striiformis f. sp. tritici in infected wheat uncovers effector functions.

The importance of wheat yellow rust disease, caused by Puccinia striiformis f. sp. tritici (Pst), has increased substantially due to the emergence of aggressive new Pst races in the last couple of decades. In an era of escalating human populations and climate change, it is vital to understand the infection mechanism of Pst in order to develop better strategies to combat wheat yellow disease. The present study focuses on the identification of small secreted proteins (SSPs) and candidate-secreted effector proteins (CSEPs) that are used by the pathogen to support infection and control disease development. We generated de novo assembled transcriptomes of Pst collected from wheat fields in central Anatolia. We inoculated both susceptible and resistant seedlings with Pst and analyzed haustoria formation. At 10 days post-inoculation (dpi), we analyzed the transcriptomes and identified 10550 Differentially Expressed Unigenes (DEGs), of which 6072 were Pst-mapped. Among those Pst-related genes, 227 were predicted as PstSSPs. In silico characterization was performed using an approach combining the transcriptomic data and data mining results to provide a reliable list to narrow down the ever-expanding repertoire of predicted effectorome. The comprehensive analysis detected 14 Differentially Expressed Small-Secreted Proteins (DESSPs) that overlapped with the genes in available literature data to serve as the best CSEPs for experimental validation. One of the CSEPs was cloned and studied to test the reliability of the presented data. Biological assays show that the randomly selected CSEP, Unigene17495 (PSTG_10917), localizes in the chloroplast and is able to suppress cell death induced by INF1 in a Nicotiana benthamiana heterologous expression system.

Proteinaceous effector discovery and characterization in filamentous plant pathogens.

The complicated interplay of plant-pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.

Identification and characterization of Dicer-like genes in leaf rust pathogen (Puccinia triticina) of wheat.

Puccinia triticina (P. triticina) is one of the most devastating fungal pathogens of wheat which causes significant annual yield loss to the crop. Understanding the gene regulatory mechanism of the biotrophic pathogen is one of the important aspects of host-pathogen interaction studies. Dicer-like genes are considered as important mediators of RNAi-based gene regulation. In this study, we report the presence of three Dicer-like genes (Pt-DCL1, Pt-DCL2, Pt-DCL3) in P. triticina genome identified through computational and biological analyses. Quantitative real-time PCR studies revealed an increase in the expression of these genes in germinating spore stages. Heterologous expression combined with mass spectrometry analysis of Pt-DCL2 confirmed the presence of a canonical Dicer-like gene in P. triticina. Phylogenetic analysis of the Pt-DCLs with the Dicer-like proteins from other organisms showed a distinct cluster of rust pathogens from the order Pucciniales. The results indicated a species-specific duplication of Dicer-like genes within the wheat rust pathogens. This study, for the first time, reports the presence of Dicer-dependent RNAi pathway in P. triticina that may play a role in gene regulatory mechanism of the pathogen during its development. Our study serves as a vital source of information for further RNAi-based molecular studies for better understanding and management of the wheat leaf rust disease.

Development of a specific AFLP-based SCAR marker for Chinese Race 34MKG of Puccinia graminis f. sp. tritici.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a fungus that causes the devastating fungalwheat stem rust disease in wheat production. Rapid identification of the physiological races of Pgt are very importance for the prevention of wheat stem rust. In this paper we developed a molecular method to identify the most prevalent race of Pgt, as a supplement for traditionally used host-specific methods. Amplified fragment length polymorphism (AFLP) was employed as a means of analyzing DNA polymorphisms in six common physiological races of Pgt in China and Ug99. In total, 64 pairs of primers were used for AFLP screening of race-specific molecular markers. One primer pair-namely, E7/M7 (5'-GACTGCGTACCAATTCG G-3'/5'-GATGAGTCCTGAGTAACGG-3')-yielded a unique band for the race 34MKG that was purified and cloned into the pGEM-T vector for sequencing. We then designed a new primer pairs (sequence-characterized amplified region marker) to amplify the 171-bp fragment and confirmed that the marker was highly specific for 34MKG. These results provide a new tool for monitoring different races of Pgt for improved control of wheat stem rust in China.