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The mimp1 element previously identified in the ascomycete fungus Fusarium oxysporum has hallmarks of miniature inverted-repeat transposable elements (MITEs): short size, terminal inverted repeats (TIRs), structural homogeneity, and a stable secondary structure. Since mimp1 has no coding capacity, its mobilization requires a transposase-encoding element. On the basis of the similarity of TIRs and target-site preference with the autonomous Tcl-like element impala, together with a correlated distribution of both elements among the Fusarium genus, we investigated the ability of mimp1 to jump upon expression of the impala transposase provided in trans. Under these conditions, we present evidence that mimp1 transposes by a cut-and-paste mechanism into TA dinucleotides, which are duplicated upon insertion. Our results also show that mimp1 reinserts very frequently in genic regions for at least one-third of the cases. We also show that the mimp1/impala double-component system is fully functional in the heterologous species F. graminearum, allowing the development of a highly efficient tool for gene tagging in filamentous fungi. [ABSTRACT FROM AUTHOR]

Background: Tunisia harbors a rich collection of unexploited durum wheat landraces ( Triticum durum ssp. durum ) that have been gradually replaced by elite cultivars since the 1970s. These landraces represent an important potential source for broadening the genetic background of elite durum wheat cultivars and for the introgression of novel genes for key traits, including disease resistance, into these cultivars. Methods: In this study, single nucleotide polymorphism (SNP) markers were used to investigate the genetic diversity and population structure of a core collection of 235 durum wheat accessions consisting mainly of landraces. The high phenotypic and genetic diversity of the fungal pathogen Pyrenophora tritici-repentis (cause of tan spot disease of wheat) in Tunisia allowed the assessment of the accessions for tan spot resistance at the adult plant stage under field conditions over three cropping seasons. A genome-wide association study (GWAS) was performed using a 90k SNP array. Results: Bayesian population structure analysis with 9191 polymorphic SNP markers classified the accessions into two groups, where groups 1 and 2 included 49.79% and 31.49% of the accessions, respectively, while the remaining 18.72% were admixtures. Principal coordinate analysis, the unweighted pair group method with arithmetic mean and the neighbor-joining method clustered the accessions into three to five groups. Analysis of molecular variance indicated that 76% of the genetic variation was among individuals and 23% was between individuals. Genome-wide association analyses identified 26 SNPs associated with tan spot resistance and explained between 8.1% to 20.2% of the phenotypic variation. The SNPs were located on chromosomes 1B (1 SNP), 2B (4 SNPs), 3A (2 SNPs), 3B (2 SNPs), 4A (2 SNPs), 4B (1 SNP), 5A (2 SNPs), 5B (4 SNPs), 6A (5 SNPs), 6B (2 SNPs), and 7B (1 SNP). Four markers, one on each of chromosomes 1B, and 5A, and two on 5B, coincided with previously reported SNPs for tan spot resistance, while the remaining SNPs were either novel markers or closely related to previously reported SNPs. Eight durum wheat accessions were identified as possible novel sources of tan spot resistance that could be introgressed into elite cultivars. Conclusion: The results highlighted the significance of chromosomes 2B, 5B, and 6A as genomic regions associated with tan spot resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Laribi, Fredua-Agyeman, Ben M’Barek, Sansaloni, Dreisigacker, Gamba, Abdedayem, Nefzaoui, Araar, Hwang, Yahyaoui and Strelkov.)

Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. A collection of P . tritici-repentis isolates from Tunisia, located in one of the main secondary centers of diversification of durum wheat, was tested for phenotypic race classification based on virulence on a host differential set and for the presence of the necrotrophic effector (NE) genes ToxA , ToxB , and toxb by PCR analysis. While races 2, 4, 5, 6, 7, and 8 were identified according to their virulence phenotypes, PCR testing indicated the presence of "atypical" isolates that induced necrosis on the wheat differential 'Glenlea,' but lacked the expected ToxA gene, suggesting the involvement of other NEs in the P . tritici-repentis /wheat interaction. Genetic diversity and the P . tritici-repentis population structure were explored further by examining 59 Tunisian isolates and 35 isolates from Algeria, Azerbaijan, Canada, Iran, and Syria using 24 simple sequence repeat markers. Average genetic diversity, overall gene flow, and percentage polymorphic loci were estimated as 0.58, 2.09, and 87%, respectively. Analysis of molecular variance showed that 81% of the genetic variance occurred within populations and 19% occurred between populations. Cluster analysis by the unweighted pair group method indicated that ToxB - isolates grouped together and were distantly related to ToxB + isolates. Based on Nei's analysis, the global collection clustered into two distinct groups according to their region of origin. The results suggest that geographic origin and the host specificity imposed by different NEs can lead to differentiation among P . tritici-repentis populations.

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., (© 2015 BSPP AND JOHN WILEY & SONS LTD.)

We identified and functionally characterized genes encoding three Galpha proteins and one Gbeta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Galpha(i) and Galpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.

With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.