Postdoctoral fellow, Washington State University
Wheat rust disease: understanding pathogen attack and plant immunity.
Wheat rust is a devastating disease of wheat that can cause over 20-80% reduction in crop yield worldwide. Efficient management of wheat rust diseases is challenging since Puccinia spp. can rapidly alter their effector repertoires in response to recognition by resistance genes. Although many putative effectors have been predicted, functional studies have been limited due to the lack of a suitable effector delivery system for wheat. Here we identified two Pseudomonas strains for delivering rust effectors. Twenty effector proteins from P. graminis and P. stiriiformis were screened and nine effectors capable of suppressing host defense responses were identified. Further, we analyzed the small RNA profiles of two wheat cultivars differentially susceptible to P. striiformis to understand gene regulation following infection. A total of 163 novel miRNAs and 182 known miRNAs were identified using 12 small RNA libraries from two cultivars - Louise and Penawawa infected with P. striiformis. 145 miRNAs targeted wheat genes, while 69 miRNAs targeted fungal genes. Of these miRNAs, 53 were differentially expressed (P &lt; 0.05) between the two infected cultivars that varied in their response to rust disease. The majority of fungal targets coded for small, secreted proteins, providing preliminary evidence for cross-kingdom control of gene regulation. Overall, this study contributes to the current repository of wheat effectors and miRNAs; and provides novel information on the yet to be characterized roles for proteins and microRNAs in the wheat-rust interactions.
Abstract: The cereal rust fungi are destructive pathogens that affect grain production worldwide. Although the genomic and transcript sequences for three Puccinia species that attack wheat have been released, the functions of large repertories of genes from Puccinia still need to be addressed to understand the infection process of these obligate parasites. Host-induced gene silencing (HIGS) has emerged a useful tool to examine the importance of rust fungus genes while growing within host plants. In this study, HIGS was used to test genes from Puccinia with transcripts enriched in haustoria for their ability to interfere with full development of the rust fungi.Approximately 1200 haustoria enriched genes from Puccinia graminis f. sp. tritici (Pgt) were identified by comparative RNA sequencing. Virus-induced gene silencing (VIGS) constructs with fragments of 86 Puccinia genes, were tested for their ability to interfere with full development of these rust fungi. Most of the genes tested had no noticeable effects, but 10 reduced Pgt development after co-inoculation with the gene VIGS constructs and Pgt. These included a predicted glycolytic enzyme, two other proteins that are probably secreted and involved in carbohydrate or sugar metabolism, a protein involved in thiazol biosynthesis, a protein involved in auxin biosynthesis, an amino acid permease, two hypothetical proteins with no conserved domains, a predicted small secreted protein and another protein predicted to be secreted with similarity to bacterial proteins involved in membrane transport. Transient silencing of four of these genes reduced development of P. striiformis (Pst), and three of also caused reduction of P. triticina (Pt) development.Partial suppression of transcripts involved in a large variety of biological processes in haustoria cells of Puccinia rusts can disrupt their development. Silencing of three genes resulted in suppression of all three rust diseases indicating that it may be possible to engineer durable resistance to multiple rust pathogens with a single gene in transgenic wheat plants for sustainable control of cereal rusts.
Pub.: 05 Aug '15, Pinned: 23 Jun '17
Abstract: Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is a costly global disease that burdens farmers with yield loss and high fungicide expenses. This sophisticated biotrophic parasite infiltrates wheat leaves and develops infection structures inside host cells, appropriating nutrients while suppressing the plant defense response. Development in most eukaryotes is regulated by small RNA molecules, and the success of host-induced gene silencing technology in Puccinia spp. implies the existence of a functional RNAi system. However, some fungi lack this capability, and small RNAs have not yet been reported in rust fungi. The objective of this study was to determine whether P. striiformis carries an endogenous small RNA repertoire.We extracted small RNA from rust-infected wheat flag leaves and performed high-throughput sequencing. Two wheat cultivars were analyzed: one is susceptible; the other displays partial high-temperature adult plant resistance. Fungal-specific reads were identified by mapping to the P. striiformis draft genome and removing reads present in uninfected control libraries. Sequencing and bioinformatics results were verified by RT-PCR. Like other RNAi-equipped fungi, P. striiformis produces large numbers of 20-22 nt sequences with a preference for uracil at the 5' position. Precise post-transcriptional processing and high accumulation of specific sRNA sequences were observed. Some predicted sRNA precursors possess a microRNA-like stem-loop secondary structure; others originate from much longer inverted repeats containing gene sequences. Finally, sRNA-target prediction algorithms were used to obtain a list of putative gene targets in both organisms. Predicted fungal target genes were enriched for kinases and small secreted proteins, while the list of wheat targets included homologs of known plant resistance genes.This work provides an inventory of small RNAs endogenous to an important plant pathogen, enabling further exploration of gene regulation on both sides of the host/parasite interaction. We conclude that small RNAs are likely to play a role in regulating the complex developmental processes involved in stripe rust pathogenicity.
Pub.: 24 Sep '15, Pinned: 23 Jun '17
Abstract: Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection the fungus secretes a number of effector proteins. Although a large repository of putative effectors have been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study we mined the available transcriptomes of Puccinia graminis and Puccinia striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector-R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS-2 and GPA/RBP-1) and one mutated R gene - Pto (Y207D). Nine out of twenty proteins, designated Shr1- Shr9 (Suppressors of Hypersensitive Response), were found to suppress HR in Nicotiana benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed non-specific hypersensitive response induced by P. syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.
Pub.: 10 Aug '16, Pinned: 23 Jun '17