Phakopsora pachyrhizi, an obligate biotrophic rust fungus, is the causal agent of Asian Soybean Rust (ASR) disease. Here, we utilized whole-genome data to explore the evolutionary patterns and population structure across 45 P. pachyrhizi isolates collected from 1972 to 2017 from diverse geographic regions worldwide. We also characterized in-silico mating-type (MAT) genes of P. pachyrhizi, in the predicted proteome of three isolates, to investigate the sexual compatibility system. Our molecular phylogenetic analysis in P. pachyrhizi inferred two distinct evolutionary lineages structured on a temporal scale, with lineage Pp1 grouping isolates obtained from 1972 to 1994, while more recently collected isolates formed a second lineage, Pp2. We found higher levels of genetic diversity in lineage Pp1, whereas lineage Pp2 exhibited a strong clonal genetic structure, with a significant lower diversity. The widespread propagation of P. pachyrhizi clonal spores across soybean-growing regions likely explains the absence of a large-scale spatial genetic structure within each lineage. Two independent isolates (TW72-1 and AU79-1) showed moderate levels of genetic admixture, suggesting potential somatic hybridization between the two P. pachyrhizi lineages. We observed no clear congruence between virulence levels of P. pachyrhizi isolates and their phylogenetic patterns. Our findings support a probable tetrapolar mating system in P. pachyrhizi. Taken together, our study offers new insights into the evolutionary history of P. pachyrhizi and demonstrates that multiple MAT genes are highly expressed during the later stages of soybean infection, suggesting their potential role in the formation of urediniospores within the life cycle of P. pachyrhizi.

Phakopsora pachyrhizi, an obligate biotrophic rust fungus, is the causal agent of Asian Soybean Rust (ASR) disease. Here, we utilized whole-genome data to explore the evolutionary patterns and population structure across 45 P. pachyrhizi isolates collected from 1972 to 2017 from diverse geographic regions worldwide. We also characterized in-silico mating-type (MAT) genes of P. pachyrhizi, in the predicted proteome of three isolates, to investigate the sexual compatibility system. Our molecular phylogenetic analysis in P. pachyrhizi inferred two distinct evolutionary lineages structured on a temporal scale, with lineage Pp1 grouping isolates obtained from 1972 to 1994, while more recently collected isolates formed a second lineage, Pp2. We found high levels of genetic diversity in lineage Pp1, whereas lineage Pp2 exhibited a strong clonal genetic structure, with a significant lower diversity. The widespread propagation of P. pachyrhizi clonal spores across soybean-growing regions likely explains the absence of a large-scale spatial genetic structure within each lineage. Two independent isolates (TW72-1 and AU79-1) showed moderate levels of genetic admixture, suggesting potential somatic hybridization between the two P. pachyrhizi lineages. We observed no clear congruence between virulence levels of P. pachyrhizi isolates and their phylogenetic patterns. Our findings support a probable tetrapolar mating system in P. pachyrhizi. Taken together, our study offers new insights into the evolutionary history of P. pachyrhizi and demonstrates that multiple MAT genes are highly expressed during the later stages of soybean infection, suggesting their potential role in the formation of urediniospores within the life cycle of P. pachyrhizi. ### Competing Interest Statement All authors declare no conflicting interest

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi , the causal agent of Asian soybean rust disease, one of the world’s most economically damaging agricultural diseases. Despite P. pachyrhizi ’s impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

ABSTRACT Asian soybean rust, caused by Phakopsora pachyrhizi , is one of the world’s most economically damaging agricultural diseases. Despite P. pachyrhizi ’s impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. We simultaneously sequenced three P. pachyrhizi genomes uncovering a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. The proliferation of TEs within the genome occurred in several bursts and correlates with the radiation and speciation of the legumes. We present data of clear de-repression of TEs that mirrors expression of virulence-related candidate effectors. We can see a unique expansion in amino acid metabolism for this fungus. Our data shows that TEs play a dominant role in P. pachyrhizi ’s genome and have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity of the genome.

Background Phakopsora pachyrhizi is a biotrophic fungal pathogen responsible for the Asian soybean rust disease causing important yield losses in tropical and subtropical soybean-producing countries. P. pachyrhizi triggers important transcriptional changes in soybean plants during infection, with several hundreds of genes being either up- or downregulated. Results Based on published transcriptomic data, we identified a predicted chitinase gene, referred to as GmCHIT1 , that was upregulated in the first hours of infection. We first confirmed this early induction and showed that this gene was expressed as early as 8 h after P. pachyrhi zi inoculation. To investigate the promoter of GmCHIT1, transgenic soybean plants expressing the green fluorescence protein (GFP) under the control of the GmCHIT1 promoter were generated. Following inoculation of these transgenic plants with P. pachyrhizi , GFP fluorescence was detected in a limited area located around appressoria, the fungal penetration structures. Fluorescence was also observed after mechanical wounding whereas no variation in fluorescence of p GmCHIT1 :GFP transgenic plants was detected after a treatment with an ethylene precursor or a methyl jasmonate analogue. Conclusion We identified a soybean chitinase promoter exhibiting an early induction by P. pachyrhizi located in the first infected soybean leaf cells. Our results on the induction of GmCHIT1 promoter by P. pachyrhizi contribute to the identification of a new pathogen inducible promoter in soybean and beyond to the development of a strategy for the Asian soybean rust disease control using biotechnological approaches.

Background: Chitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals (mainly invertebrates), several protists and a few algae, playing an essential role in the development of many of them. This polysaccharide is produced by type 2 glycosyltransferases, called chitin synthases (CHS). There are several contradictory classifications of CHS isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate.Results: A genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about 130 genomes. Phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences (e.g. highly variable regions, truncated or recombined sequences, long-branch attraction). This allowed us to revise and unify the fungal CHS classification and to study the evolutionary history of the CHS multigenic family. This update has the advantage of being user-friendly due to the development of a dedicated website (http://wwwabi.snv.jussieu.fr/public/CHSdb), and it includes any correspondences with previously published classifications and mutants. Concerning the evolutionary history of CHS, this family has mainly evolved via duplications and losses. However, it is likely that several horizontal gene transfers (HGT) also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria.Conclusions: This comprehensive multi-species analysis contributes to the classification of fungal CHS, in particular by optimizing its robustness, consensuality and accessibility. It also highlights the importance of HGT in the evolutionary history of CHS and describes bacterial chs genes for the first time. Many of the bacteria that have acquired a chitin synthase are plant pathogens (e.g. Dickeya spp; Pectobacterium spp; Brenneria spp; Agrobacterium vitis and Pseudomonas cichorii). Whether they are able to produce a chitin exopolysaccharide or secrete chitooligosaccharides requires further investigation.

Fosetyl-Al is a well-known anti-oomycetes fungicide with efficacy based on an indirect action as stimulation of the natural plant defenses. Field phenotypic observations in apple, pear and cherry orchards have revealed very positive effect of fosetyl-Al on flowering the year following treatment. An advantageous effect was noticed both on quantity as on the quality of flower buds. Flower cluster of trees treated with Aliette in the previous season had more flowers and a tendency of being surrounded by more well developed rosette leaves. Fosetyl-Al's mode of action analysis was conducted in parallel and independently on the vine-downy mildew patho-system to elucidate the effect as stimulator of natural plant defense. Transcriptomic analysis clearly showed involvement of fosetyl-Al in defense response with genes deregulation in chalcone synthase biosynthetic pathway and in stilbenes accumulation. Those pathways are the most frequently and best characterized defense mechanisms described in vine. Besides that, 3 clusters of genes corresponding to auxine responsive proteins were found slightly up-regulated with fosetyl-Al preventative treatment. Speculation on the role of those genes induced by fosetyl-Al and consequently the input on flowering quality on apples and pears could be discussed.

This chapter focuses on synthetic chemicals that have been defined as host defense inducers by the Fungicide Resistance Action Committee (FRAC) as code P1, such as acibenzolar-S-methyl (ASM); code P2, such as probenazole (PBZ); and code P3, such as tiadinil and isotianil. According to the inducing agent involved and the subsequent molecular mechanism, two major types of induced resistance have been identified: systemic acquired resistance (SAR) and induced systemic resistance (ISR). Transcription factors differentially regulate a number of genes involved in defense, leading ultimately to the production of defense proteins. In SAR expressing plants, the defense response occurs rapidly and/or efficiently on pathogen challenge. Currently, PBZ is mainly used as combination granule formulations with different insecticides for nursery box application in Japan, providing long-lasting control against a variety of pests in rice. Isotianil, by itself, does not exhibit any activity against pathogens but rather protects plants against infection when applied at an early developmental stage.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

The expanding field of fungal genomics stimulates the development of genome wide functional tools and comparative analyses in plant pathogenic fungi. As a consequence, transcriptomic, proteomic and metabolomic studies coupled with high throughput forward and reverse genetics are now available in a significant number of fungal plant pathogens (e.g. Ustilago maydis, Magnaporthe grisea, Fusarium graminea rum, Botrytis cinerea). Genomics together with classical biochemical tools and microscopy offer the possibility to accelerate the identification of the biochemical mode of action of novel fungicides. This knowledge is also required to discover efficiently novel antifungal compounds and to characterize and follow efficiently the emergence of resistance. The available genomic tools for plant pathogenic fungi will be reviewed as exemplified with the mode of action of fluopicolide, a novel fungicide active against Oomycetes. Biological studies performed with Phytophthora infestans and Plasmopara viticola showed that fluopicolide affects the release and motility of zoospores and the germination of cysts, as well as mycelial growth and sporulation. Biochemical studies showed that its mode of action differs from that of known anti-oomycetes compounds. Fluopicolide does not show cross-resistance to commercial fungicide classes such as phenylamides, strobilurins (QoIs) and carboxylic acid amides (CAAs). Cytological studies in P infestans showed that fluopicolide specifically modifies the spatial and cellular distribution of proteins labelled by antibodies specific for animal cytoskeleton associated proteins spectrin. Treatments with fluopicolide induced a fast redistribution of spectrin-like protein(s) from the membrane to the cytoplasm in both hyphae and zoospores. Whereas animal spectrin(s) play an important role in membrane stability, they are poorly characterized in fungi and oomycetes. Cytoskeletal proteins such as actins, tubulins, integrins and spectrins provide structural stability to cells as they form a network sustaining the plasma membrane. Fluopicolide may interfere and destabilize this network leading to cell disorganization. This hypothesis is supported by the observation that treatments of zoospores lead to the relocalization of spectrin-like protein(s) into the cytoplasm within a few minutes followed immediately by cell swelling and burst. Preliminary data of gene expression profiling in P. sojae treated cells showed a differential expression (up and down regulation) of genes involved in vesicular transport. The link between golgi function, vesicle transport and cellular relocation of spectrin like proteins will be discussed.

Background Tetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea , Botrytis cinerea and Colletotrichum lindemuthianum , and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor , and also their functional analysis in M. grisea . Results The exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2 , which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor . A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea , suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Δtsp3 and Δtpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Δpls1 mutants, which are completely non-pathogenic on barley and rice. Conclusion A new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea . The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.

Field strains of Plasmopara viticola, the causal agent of grapevine downy mildew, were characterised for their sensitivity to QoI (Quinol outside Inhibitors) fungicides. At the gene level, resistance to this agricultural fungicide class is due to a Single Nucleotide Polymorphism on cytochrome bc1 mitochondrial gene causing an amino acid change (Glycine to Alanine at position 143 of the gene) in an enzyme of the respiratory chain. The mutation can be detected on mitochondrial DNA fragments with quantitative PCR amplification using allele specific primers and a Sybr Green detection method. A comparative analysis of this quantitative PCR measurement of mutant allele frequencies and the determination of biological phenotypes of several P. viticola samples has detected the presence of two characterised population classes: samples sensitive to QoI fungicides containing less than 0.1% of mutant allele and resistant ones containing more than 2%. The samples in which mutant allele percentage is between these two limits are mostly sensitive but, under the growth conditions used, they are able to become resistant and vice versa. Thus it seems that in a window between 0.1% to 2% of mutant allele present in the sample tested, the resistance of P. viticola to QoI fungicides is unstable.