A small secreted protein from Zymoseptoria tritici interacts with a wheat E3 ubiquitin ligase to promote disease.
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ABSTRACT:
SUBMITTER: Karki SJ
PROVIDER: S-EPMC7853600 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mycosphaerella graminicola (Zymoseptoria tritici commonly known as Septoria), the causal agent of Septoria Leaf Blotch (STB), is considered one of the major threats to European wheat production. Previous studies have shown the importance of ubiquitination in plant defence against a multitude of pathogens. However the ubiquitination machinery in wheat is under studied, particularly E2 enzymes that have the ability to control the ubiquitination and thereby the fate of many different target proteins. In this study we identify an E2 enzyme, Triticum aestivum Ubiquitin conjugating enzyme 4 (TaU4) that functions in wheat defence against Septoria. We demonstrate TaU4 to be a bona fide E2 enzyme through an E2 charging assay. TaU4 localises in both the cytoplasm and nucleus, therefore potentially interacting with E3 ligases and substrate proteins in multiple compartments. Virus Induced Gene Silencing of TaU4 in wheat leaves resulted in delayed development of disease symptoms, reduced Septoria growth and reproduction. We conclude that TaU4 is a novel negative regulator of defence against Septoria.
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Project description:Cross-kingdom RNA interference (RNAi) has been shown to play important roles during plant-pathogen interactions, and both plants and pathogens can use small RNAs (sRNAs) to silence genes in each other. This bidirectional cross-kingdom RNAi was still unexplored in the wheat-Zymoseptoria tritici pathosystem. Here, we performed a detailed analysis of the sRNA bidirectional crosstalk between wheat and Z. tritici. Using a combination of small RNA sequencing (sRNA-seq) and microRNA sequencing (mRNA-seq), we were able to identify known and novel sRNAs and study their expression and their action on putative targets in both wheat and Z. tritici. We predicted the target genes of all the sRNAs in either wheat or Z. tritici transcriptome and used degradome analysis to validate the cleavage of these gene transcripts. We could not find any clear evidence of a cross-kingdom RNAi acting by mRNA cleavage in this pathosystem. We also found that the fungal sRNA enrichment was lower in planta than during in vitro growth, probably due to the lower expression of the only Dicer gene of the fungus during plant infection. Our results support the recent finding that Z. tritici sRNAs cannot play important roles during wheat infection. However, we also found that the fungal infection induced wheat sRNAs regulating the expression of specific wheat genes, including auxin-related genes, as an immune response. These results indicate a role of sRNAs in the regulation of wheat defenses during Z. tritici infection. Our findings contribute to improve our understanding of the interactions between wheat and Z. tritici.
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Project description:The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.
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Project description:Cell death processes in eukaryotes shape normal development and responses to the environment. For plant-microbe interactions, initiation of host cell death plays an important role in determining disease outcomes. Cell death pathways are frequently initiated following detection of pathogen-derived molecules which can lead to resistance or susceptibility to disease depending on pathogen lifestyle. We previously identified several small secreted proteins (SSPs) from the wheat-infecting fungus Zymoseptoria tritici that induce rapid cell death in Nicotiana benthamiana following Agrobacterium-mediated delivery and expression (agroinfiltration). Here we investigated whether the execution of host cells was mechanistically similar in response to different Z. tritici SSPs. Using RNA sequencing, we found that transient expression of four Z. tritici SSPs led to massive transcriptional reprogramming within 48 h of agroinfiltration. We observed that distinct host gene expression profiles were induced dependent on whether cell death occurs in a cell surface immune receptor-dependent or -independent manner. These gene expression profiles involved differential transcriptional networks mediated by WRKY, NAC and MYB transcription factors. In addition, differential expression of genes belonging to different classes of receptor-like proteins and receptor-like kinases was observed. These data suggest that different Z. tritici SSPs trigger differential transcriptional reprogramming in plant cells.
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Project description:The fungus Zymoseptoria tritici causes Septoria tritici blotch of wheat. Pathogenicity begins with spore germination, followed by stomata invasion by hyphae, mesophyll colonization and fruiting body formation. It was previously found that entry into the plant via stomata occurs in a non-synchronized way over several days, while later developmental steps, such as early and late fruiting body formation, were reported to follow each other in time. This suggests synchronization of the pathogen population in planta prior to sporulation. Here, we image a fluorescent Z. tritici IPO323-derived strain during infection. We describe 6 morphologically distinct developmental stages, and determine their abundance in infected leaves, with time post inoculation. This demonstrates that 3-5 stages co-exist in infected tissues at any given time. Thus, later stages of pathogen development also occur asynchronously amongst the population of infecting cells. This merits consideration when interpreting transcriptomics or proteomics data gathered from infected plants.
| S-EPMC7812371 | biostudies-literature
Project description:Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.
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Project description:Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.
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Project description:Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and Spry2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The Spry2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser(112) and Ser(121), on Spry2. The phospho-Ser(112/121) region on Spry2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate Spry2 via Lys(48) on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular Spry2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in Spry2 levels were prevented by Spry2-specific small interfering RNA. Mnk2 silencing decreased Spry2-Nedd4 interactions and also augmented the ability of Spry2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.
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Project description:This paper reviews current knowledge about genes for resistance to Septoria tritici blotch (STB) of wheat, caused by Zymoseptoria tritici (formerly Mycosphaerella graminicola). These genes can be placed into two classes, although a few may have characteristics of both classes. Qualitative resistance is controlled by genes which control large fractions of genetic variation, 21 of which have been discovered and mapped so far. Most of them have been shown to be genotype-specific, being effective against the minority of Z. tritici isolates which are avirulent, and Stb6 has been shown to control a gene-for-gene relationship. Most qualitative resistances are unlikely to be durable and some formerly effective genes have been overcome by the evolution of pathogen virulence. Quantitative resistance is generally controlled by genes with small-to-moderate effects on STB. They have generally weaker specificity than qualitative genes and have provided more durable resistance. 89 genome regions carrying quantitative trait loci (QTL) or meta-QTL have been identified to date. Some QTL have been mapped at or near loci of qualitative genes, especially Stb6, which is present in several sources of resistance. Another gene of particular interest is Stb16q, which has been effective against all Z. tritici isolates tested so far. In addition to resistance, the susceptibility of wheat cultivars to STB can also be reduced by disease escape traits, some of which may be undesirable in breeding. The fundamental requirements for breeding for STB-resistance are genetic diversity for resistance in wheat germplasm and a field trial site at which STB epidemics occur regularly and effective selection can be conducted for resistance combined with other desirable traits. If these are in place, knowledge of resistance genes can be applied to improving control of STB.
| S-EPMC4510316 | biostudies-literature
Project description:Development of novel strategies to control fungal plant pathogens requires understanding of their cellular organisation and biology. Live cell imaging of fluorescent organelle markers has provided valuable insight into various aspects of their cell biology, including invasion strategies in plant pathogenic fungi. Here, we introduce a set of 17 vectors that encode fluorescent markers to visualize the plasma membrane, endoplasmic reticulum (ER), chromosomes, the actin cytoskeleton, peroxisomes and autophagosomes in the wheat pathogen Zymoseptoria tritici. We fused either enhanced green-fluorescent protein (eGFP) or a codon-optimised version of GFP (ZtGFP) to homologues of a plasma membrane-located Sso1-like syntaxin, an ER signalling and retention peptide, a histone H1 homologue, the LifeAct actin-binding peptide, a mitochondrial acetyl-CoA dehydrogenase, a peroxisomal import signal and a homologue of the ubiquitin-like autophagosomal protein Atg8. We expressed these markers in wildtype strain IPO323 and confirmed the specificity of these markers by counterstaining or physiological experiments. This new set of molecular tools will help understanding the cell biology of the wheat pathogen Z. tritici.
| S-EPMC5536155 | biostudies-literature