Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance.
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SUBMITTER: Lee WS
PROVIDER: S-EPMC4510315 | biostudies-literature | 2015 Jun
REPOSITORIES: biostudies-literature
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Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance.
Lee Wing-Sham WS Rudd Jason J JJ Kanyuka Kostya K
Fungal genetics and biology : FG & B 20150601
Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious ...[more]
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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:BackgroundSeptoria tritici blotch (STB), caused by Zymoseptoria tritici (Z. tritici), is an important biotic threat to durum wheat in the entire Mediterranean Basin. Although most durum wheat cultivars are susceptible to Z. tritici, research in STB resistance in durum wheat has been limited.ResultsIn our study, we have identified resistance to a wide array of Z. tritici isolates in the Tunisian durum wheat landrace accession 'Agili39'. Subsequently, a recombinant inbred population was developed and tested under greenhouse conditions at the seedling stage with eight Z. tritici isolates and for five years under field conditions with three Z. tritici isolates. Mapping of quantitative trait loci (QTL) resulted in the identification of two major QTL on chromosome 2B designated as Qstb2B_1 and Qstb2B_2. The Qstb2B_1 QTL was mapped at the seedling and the adult plant stage (highest LOD 33.9, explained variance 61.6%), conferring an effective resistance against five Z. tritici isolates. The Qstb2B_2 conferred adult plant resistance (highest LOD 32.9, explained variance 42%) and has been effective at the field trials against two Z. tritici isolates. The physical positions of the flanking markers linked to Qstb2B_1 and Qstb2B_2 indicate that these two QTL are 5 Mb apart. In addition, we identified two minor QTL on chromosomes 1A (Qstb1A) and chromosome 7A (Qstb7A) (highest LODs 4.6 and 4.0, and explained variances of 16% and 9%, respectively) that were specific to three and one Z. tritici isolates, respectively. All identified QTL were derived from the landrace accession Agili39 that represents a valuable source for STB resistance in durum wheat.ConclusionThis study demonstrates that Z. tritici resistance in the 'Agili39' landrace accession is controlled by two minor and two major QTL acting in an additive mode. We also provide evidence that the broad efficacy of the resistance to STB in 'Agili 39' is due to a natural pyramiding of these QTL. A sustainable use of this Z. tritici resistance source and a positive selection of the linked markers to the identified QTL will greatly support effective breeding for Z. tritici resistance in durum wheat.
| S-EPMC9112612 | biostudies-literature
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.
| S-EPMC6638447 | biostudies-literature
Project description:The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential. IMPORTANCE Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici, have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides' efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici. The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici.
| S-EPMC5656749 | biostudies-literature
Project description:BackgroundWheat is one of the world's most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars.ResultsEighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar.ConclusionThese results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.
| S-EPMC10996218 | biostudies-literature
Project description:Zymoseptoria tritici is a globally distributed fungal pathogen which causes Septoria tritici blotch on wheat. Management of the disease is attempted through the deployment of resistant wheat cultivars and the application of fungicides. However, fungicide resistance is commonly observed in Z. tritici populations, and continuous monitoring is required to detect breakdowns in fungicide efficacy. We recently reported azole-resistant isolates in Australia; however, it remained unknown whether resistance was brought into the continent through gene flow or whether resistance emerged independently. To address this question, we screened 43 isolates across five Australian locations for azole sensitivity and performed whole-genome sequencing on 58 isolates from seven locations to determine the genetic basis of resistance. Population genomic analyses showed extremely strong differentiation between the Australian population recovered after azoles began to be used and both Australian populations recovered before azoles began to be used and populations on different continents. The apparent absence of recent gene flow between Australia and other continents suggests that azole fungicide resistance has evolved de novo and subsequently spread within Tasmania. Despite the isolates being distinct at the whole-genome level, we observed combinations of nonsynonymous substitutions at the CYP51 locus identical to those observed elsewhere in the world. We observed nine previously reported nonsynonymous mutations as well as isolates that carried a combination of the previously reported L50S, S188N, A379G, I381V, Y459DEL, G460DEL, and N513K substitutions. Assays for the 50% effective concentration against a subset of isolates exposed to the tebuconazole and epoxiconazole fungicides showed high levels of azole resistance. The rapid, parallel evolution of a complex CYP51 haplotype that matches a dominant European haplotype demonstrates the enormous potential for de novo resistance emergence in pathogenic fungi.IMPORTANCE Fungicides are essential to control diseases in agriculture because many crops are highly susceptible to pathogens. However, many pathogens rapidly evolve resistance to fungicides. A large body of studies have described specific mutations conferring resistance and have often made inferences about the origins of resistance based on sequencing data from the target gene alone. Here, we show the de novo acquisition of resistance to the ubiquitously used azole fungicides in genetically isolated populations of the wheat pathogen Zymoseptoria tritici in Tasmania, Australia. We confirm evidence for parallel evolution through genome-scale analyses of representative worldwide populations. The emergence of complex resistance haplotypes following a well-documented recent introduction of azoles into Australian farming practices demonstrates how rapidly chemical resistance evolves in agricultural ecosystems.
| S-EPMC6365823 | 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
Project description:Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host-pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z.?tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild-type phenotypes. In?planta expression analyses showed that ZtWor1 is up-regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z.?tritici wild-type strain and the ZtWor1 mutant, which was particularly expressed in?planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the ?-subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock-out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens.
| S-EPMC6638687 | biostudies-literature
Project description:An association genetics analysis was conducted to investigate the genetics of resistance to Septoria tritici blotch, caused by the fungus Zymoseptoria tritici (alternatively Mycosphaerella graminicola), in cultivars and breeding lines of wheat (Triticum aestivum) used in the UK between 1860 and 2000. The population was tested with Diversity Array Technology (DArT) and simple-sequence repeat (SSR or microsatellite) markers. The lines formed a single population with no evidence for subdivision, because there were several common ancestors of large parts of the pedigree. Quantitative trait loci (QTLs) controlling Septoria resistance were postulated on 11 chromosomes, but 38% of variation was not explained by the identified QTLs. Calculation of best linear unbiased predictions (BLUPs) identified lineages of spring and winter wheat carrying different alleles for resistance and susceptibility. Abundant variation in Septoria resistance may be exploited by crossing well-adapted cultivars in different lineages to achieve transgressive segregation and thus breed for potentially durable quantitative resistance, whereas phenotypic selection for polygenic quantitative resistance should be effective in breeding cultivars with increased resistance. The most potent allele reducing susceptibility to Septoria, on chromosome arm 6AL, was associated with reduced leaf size. Genes which increase susceptibility to Septoria may have been introduced inadvertently into UK wheat breeding programmes from cultivars used to increase yield, rust resistance and eyespot resistance between the 1950s and 1980s. This indicates the need to consider trade-offs in plant breeding when numerous traits are important and to be cautious about the use of non-adapted germplasm.
| S-EPMC5297993 | biostudies-literature