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Multiple alleles were constantly detected in Alternaria solani isolates by simple sequence repeat (SSR) analysis, and sectors were also observed in their subcultures. These preliminary results and observations point to a possible parasexual cycle in A. solani. In this study, codominant SSR markers were used as molecular markers on the chromosomes of A. solani and single-conidium subculture was used to simulate the mitosis process of A. solani in nature. The number of alleles at locus As-95236 changed from 2 to 1 as a molecular marker for haploidy of parasexuality of A. solani. Fifty monosporic F1 strains were tested. The results showed that two parent strains lost allele with a haploid probability of 38%. For F2 strains, the results showed that all four F1 strains lost allele with a haploid probability of 75%. Since sexual recombination of A. solani has not been found so far, the allele lost in the subcultures of A. solani isolates provides molecular evidence for the existence of parasexual reproduction in A. solani.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi. Supplementary Information The online version contains supplementary material available at 10.1007/s00294-021-01157-4.

Aspergillus fumigatusis a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical forA. fumigatusto adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply inA. fumigatusdue to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis ofA. fumigatusisolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799A. fumigatusisolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acuteAspergillusinfection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allowsA. fumigatusto adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology ofA. fumigatusin the human lung.

Aspergillus nidulans is a fungal model organism extensively used in genetic approaches. It may reproduce sexually and asexually, with a well-defined parasexual cycle. The current paper demonstrates that the limitation of nitrogen source facilitates the production of A. nidulans's nonmeiotic recombinants directly from heterokaryons, without the recovery of the diploid phase. Heterokaryons formed between master strains were inoculated in sodium nitrate-low (basal medium [BM]) and sodium nitrate-rich media (minimal medium [MM]). All mitotic segregants produced by the heterokaryons were tested for their mitotic stability in the presence of benomyl, the haploidizing agent. Only mitotically stable haploid segregants were selected for subsequent analysis. Phenotypic analyses of such haploids favored the characterization of nonmeiotic recombinants. As the number of such recombinants was higher in BM than in MM, nitrogen limitation may have facilitated the isolation of nonmeiotic recombinants from heterokaryons by stimulating nuclear fusion still inside the heterokaryotic mycelium as a survival strategy.

Giardia duodenalis captured the attention of Leeuwenhoek in 1681 while he was examining his own diarrheal stool, but, ironically, it did not really gain attention as a human pathogen until the 1960s, when outbreaks were reported. Key technological advances, including in vitro cultivation, genomic and proteomic databases, and advances in microscopic and molecular approaches, have led to an understanding that this is a eukaryotic organism with a reduced genome rather than a truly premitochondriate eukaryote. This has included the discovery of mitosomes (vestiges of mitochondria), a transport system with many of the features of the Golgi apparatus, and even evidence for a sexual or parasexual cycle. Cell biology approaches have led to a better understanding of how Giardia survives with two nuclei and how it goes through its life cycle as a noninvasive organism in the hostile environment of the lumen of the host intestine. Studies of its immunology and pathogenesis have moved past the general understanding of the importance of the antibody response in controlling infection to determining the key role of the Th17 response. This work has led to understanding of the requirement for a balanced host immune response that avoids the extremes of an excessive response with collateral damage or one that is unable to clear the organism. This understanding is especially important in view of the remarkable ranges of early manifestations, which range from asymptomatic to persistent diarrhea and weight loss, and longer-term sequelae that include growth stunting in children who had no obvious symptoms and a high frequency of postinfectious irritable bowel syndrome (IBS).

Adaptive radiations fuel speciation and are characterized by rapid genetic diversification and expansion into new ecological niches. Historically, these processes were believed to be driven by selection on novel mutations but genomic analyses now indicate that standing variation and gene flow often have prominent roles. How “old” variation is combined, however, and its resulting genetic architecture within newly-adapted populations is not well understood. We reconstructed a recent radiation in the fungus, Pyricularia oryzae, that spawned a population pathogenic to eleven grass genera, and caused two new plant diseases: wheat blast - already a serious threat to global agriculture - and gray leaf spot of ryegrasses. We show that the new population evolved in a multi-hybrid swarm using only the standing variation that was present in seven individuals from five distinct, host-specialized lineages. Sexual and parasexual recombination within the swarm reassorted key host-specificity factors and generated more diversity in possibly just a few weeks than existing lineages had accumulated over hundreds to thousands of years. We suggest that the process was initiated by sexual opportunity arising when a fertile fungal strain was imported into Brazil on Urochloa introduced as forage for beef production; and we further contend that the host range expansion was largely fortuitous, with host selection playing little, if any, role in driving the process. Finally, we believe that our findings point to an overlooked role for happenstance in creating situations that allow organisms to skirt rules that would normally hold evolution in check.Commercial Endorsement DisclaimerMention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.Equal Opportunity/Non-Discrimination StatementUSDA is an equal opportunity provider and employer.

Candida tropicalis is one of the most common opportunistic yeast pathogens of humans, especially prevalent in tropical and subtropical regions. This yeast has broad ecological distributions, can be found in both terrestrial and aquatic ecosystems, including being associated with a diversity of trees, animals, and humans. Evolutionary theory predicts that organisms thriving in diverse ecological niches likely have efficient mechanisms to generate genetic diversity in nature. Indeed, abundant genetic variations have been reported in natural populations (both environmental and clinical) of C. tropicalis. However, at present, our understanding on how genetic diversity is generated in natural C. tropicalis population remains controversial. In this paper, I review the current understanding on the potential modes of reproduction in C. tropicalis. I describe expectations of the three modes of reproduction (sexual, parasexual, and asexual) and compare them with the observed genotypic variations in natural populations. Though sexual and parasexual reproduction cannot be excluded, the analyses suggest asexual reproduction alone could explain all the observations reported so far. The results here have implications for understanding the evolution and epidemiology of C. tropicalis and other related human fungal pathogens.

Anastomosis of conidia or hyphae is the first step towards the establishment of heterokaryons and the parasexual cycle in ascomycetes. This process, however, is often hampered in natural populations of fungi by vegetative or heterokaryon incompatibility systems. The asexual plant pathogenic ascomycetes of the genus Verticillium are characterized by great ecological and economic worldwide significance. Based on complementation testing of nit mutants (in the nitrate assimilation pathway), strains of these fungi are classified to Vegetative Compatibility Groups (VCGs), which are traditionally considered to comprise genetically homogeneous and isolated subspecific groups. The goal of this work was to study heterokaryosis/heterokaryon incompatibility mainly in V. dahliae with population, genetic, cell biology, genomic and molecular-functional experimental approaches. With these methods, (i) new molecular tools were developed for the rapid and accurate molecular prediction of V. dahliae VCGs, (ii) a new methodology was developed for the nit mutants-based high-throughput VCG classification of fungal strains, (iii) it was found that V. dahliae VCGs are not genetically homogeneous and absolute genetic barriers are absent between VCGs, (iv) a marked impact of the genetic method and the markers used on the VCG classification of strains was determined, (v) fusion of conidia via conidial anastomosis tubes was studied under different environmental conditions, and it was shown that nuclei can migrate through anastomosis bridges in Verticillium, resulting in the formation of heterokaryons, (vi) it was found that hyphae of incompatible V. dahliae strains exhibit a mutual tolerance behavior leading to incompatible anastomoses and heterokaryons, which are then rapidly isolated and led to cell death, (vii) Verticillium homologs of known heterokaryon incompatibility genes were identified and all genes of these fungi with putatively relevant domains were identified and in silico analyzed, (viii) efficient methods for the genetic transformation of V. dahliae were optimized and employed for the functional study of het-c1, het-c2, un-24, vib-1, hex-1 and MAT1-2-1 genes through the construction of gene knock-out deletion mutants; several phenotypic alterations both in the compatibility behavior and fundamental morphogenetic/physiological processes were recorded.%%%%Ο παραφυλeτικός κύκλος στους ασκομύκητeς ξeκινά μe την αναστόμωση σπορίων ή υφών διαφορeτικών γονοτύπων προς σχηματισμό eτeροκαρύων, ο οποίος ωστόσο συχνά παρeμποδίζeται στους φυσικούς πληθυσμούς από συστήματα eτeροκαρυωτικής ασυμβατότητας. Οι αφυλeτικοί φυτοπαθογόνοι μύκητeς του γένους Verticillium έχουν μeγάλη οικολογική και οικονομική σημασία διeθνώς. Βάσeι της μeθόδου συμπληρωματικότητας μeταλλαγών nit (στο μονοπάτι αφομοίωσης των νιτρικών ιόντων) τα στeλέχη των μυκήτων κατατάσσονται σe Ομάδeς Βλαστητικής Συμβατότητας (Vegetative Compatibility Groups, VCGs), οι οποίeς θeωρeίται παραδοσιακά πως αποτeλούν γeνeτικά ομοιογeνeίς και απομονωμένeς eνδοeιδικές υποομάδeς. Ο στόχος της…

Background Asexual fungi include important pathogens of plants and other organisms, and their effective management requires understanding of their evolutionary dynamics. Genetic recombination is critical for adaptability and could be achieved via heterokaryosis — the co-existence of genetically different nuclei in a cell resulting from fusion of non-self spores or hyphae — and the parasexual cycle in the absence of sexual reproduction. Fusion between different strains and establishment of viable heterokaryons are believed to be rare due to non-self recognition systems. Here, we investigate the extent and mechanisms of cell fusion and heterokaryosis in the important asexual plant pathogen Verticillium dahliae. Results We used live-cell imaging and genetic complementation assays of tagged V. dahliae strains to analyze the extent of non-self vegetative fusion, heterokaryotic cell fate, and nuclear behavior. An efficient CRISPR/Cas9-mediated system was developed to investigate the involvement of autophagy in heterokaryosis. Under starvation, non-self fusion of germinating spores occurs frequently regardless of the previously assessed vegetative compatibility of the partners. Supposedly “incompatible” fusions often establish viable heterokaryotic cells and mosaic mycelia, where nuclei can engage in fusion or transfer of genetic material. The molecular machinery of autophagy has a protective function against the destruction of “incompatible” heterokaryons. Conclusions We demonstrate an imperfect function of somatic incompatibility systems in V. dahliae. These systems frequently tolerate the establishment of heterokaryons and potentially the initiation of the parasexual cycle even between strains that were previously regarded as “incompatible.”

BackgroundThe kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica’s digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss.Methodology/Principal FindingsThe genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units.Conclusion/SignificanceThese large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.Author SummaryCutaneous leishmaniasis is mainly caused by Leishmania tropica in the Middle East, where it is known for treatment failure and a need for prolonged and/or multiple treatments. Several factors affect the clinical presentation and treatment outcome, such as host genetic variability and specific immune response, as well as environmental factors and the vector species. Little is known about the parasite genome and its influence on treatment response. By analysing the genome of 22 isolates of L. tropica, we have revealed extensive genomic variation and a complex population structure with evidence of genetic exchange within and among the isolates, indicating a possible presence of sexual or parasexual mechanisms. Understanding the Leishmania genome better may improve future treatment and better understanding of treatment failure and relapse.

A survey of Acanthamoeba in 100 public freshwater sources in 28 provinces across Thailand has identified 9 genotypes comprising T2/6, T3-T5, T9, T11, T12, T18 and a novel ‘T23’ among 131 isolates. Sequencing of the near complete 18S rRNA gene of Acanthamoeba of all isolates has shown that the most predominant genotype T4 found in 87 isolates (66.4%) contained 4 subtypes, i.e. T4A, T4B, T4C and T4F, while all isolates assigned to genotype T2/6 belonged to subtype B. Among intron-bearing genotypes, most isolates harbouring genotype T3 contained S516 introns, characterised by 3 distinct variants whilst all genotypes T4A and T5 were intronless. Identical 18S rRNA sequences of Acanthamoeba were identified across regions of the country and four isolates in this study shared the same sequences with those from remote nations, suggesting that some strains have reproductive success in diverse ecological niche. Nucleotide diversity of genotypes T2/6B, T3, T4, T9 and T11 in this study was significantly less than that among global isolates outside Thailand, implying that limited sequence diversity occurred within local populations. A remarkably higher level of nucleotide diversity in genotype T11 than those of other genotypes (0.041 vs. 0.012–0.024) could be due to cryptic subtypes. Recombination breakpoints have been detected within genotypes and subtypes as well as within isolates despite no evidence for sexual and parasexual cycles in the genus Acanthamoeba. Tajima’s D, Fu & Li’s D* and F* statistics revealed significantly negative deviation from neutrality across genotypes and subtypes, implying purifying selection in this locus. The 18S rRNA gene of the novel genotype ‘T23’ displayed 7.82% to 28.44% sequence differences in comparison with all known genotypes. Both Bayesian and maximum likelihood phylogenetic trees have placed genotype T23 as sister to the clade comprising genotypes T10, T12 and T14, all of these possess cyst structure belonging to morphological group III. Hence, Acanthamoeba bangkokensis sp. nov. is proposed for this novel genotype. It is likely that more genotypes of Acanthamoeba remain to be discovered while the evolution of the 18S rRNA gene of this pathogenic-free living amoeba seems to be ongoing.

Fungi in the genus Metarhizium are soil-borne plant–root endophytes and rhizosphere colonizers, but also potent insect pathogens with highly variable host ranges. These ascomycete fungi are predominantly asexually reproducing and ancestrally haploid, but two independent origins of persistent diploidy within the Coleoptera-infecting Metarhizium majus species complex are known and has been attributed to incomplete chromosomal segregation following meiosis during the sexual cycle. There is also evidence for infrequent sexual cycles in the locust-specific pathogenic fungus Metarhizium acridum (Hypocreales: Clavicipitaceae), which is an important entomopathogenic biocontrol agent used for the control of grasshoppers in agricultural systems as an alternative to chemical control. Here, we show that the genome of the M. acridum isolate ARSEF 324, which is formulated and commercially utilized is functionally diploid. We used single-molecule real-time sequencing technology to complete a high-quality assembly of ARSEF 324. K-mer frequencies, intragenomic collinearity between contigs and single nucleotide variant read depths across the genome revealed the first incidence of diploidy described within the species M. acridum. The haploid assembly of 44.7 Mb consisted of 20.8% repetitive elements, which is the highest proportion described of any Metarhizium species. The long-read diploid genome assembly sheds light on past research on this strain, such as unusual high UVB tolerance. The data presented here could fuel future investigation into the fitness landscape of fungi with infrequent sexual reproduction and aberrant ploidy levels, not least in the context of biocontrol agents.

The genetic-exchange process of some endophytic root fungi whose teleomorphs have not been discovered is unknown. To assess parasexuality in the root-colonizing fungus Glutinomyces brunneus, we investigated the transmission of genetic markers in this fungus. We generated strains resistant to benomyl and hygromycin B and performed co-cultivation; dual resistance was used as a selection marker for genetic exchange between hyphae. Co-cultivation of benomyl- and hygromycin B-resistant strains yielded significantly larger numbers of dual-resistant colonies than did co-cultivation of strains resistant to the same agent, which suggests genetic exchange between the two vegetative hyphae. This was not simply due to heterokaryosis because all dual-resistant strains were monokaryotic. Also, half of the analyzed dual-resistant strains were homozygous in β-tubulin gene which is associated with benomyl resistance; thus, their simple diploidization was rejected for these strains. During selective cultivation, temporal hyphal growth occurred from the point at which two hyphae touched, which stopped after 2 weeks; this was followed by growth of true dual-resistant hyphae. Transmission of resistance markers was more frequent during co-cultivation on 2% malt extract agar than on other media, which suggests a role of medium composition. Genetic exchange between resistant strains that did not share an ancestor was limited, which implies vegetative incompatibility in this fungus. Our findings suggest the partial presence of parasexual potential in G. brunneus.

Blast of rice caused by Pyricularia grisea is one of the most devastating diseases of rice. Because of importance of the disease and the fact that pyricularia grisea is considered to be notorious and model species. The variability in cultural characteristics of fifty isolates of P. grisea were taken from different regions of Jharkhand state. Out of fifty isolates of P. grisea, colony color of six isolates were found to be as greyish white color, three isolates were blackish grey, six isolates as white color, three isolates as whitish grey color, five isolates were whitish black and twenty seven isolates were recorded as blackish white in color. The growth pattern of 47 isolates of P. grisea showed circular growth pattern and three isolates have irregular growth pattern but elevation of the mycelium differs from flat to raised. Out of fifty isolates of P. grisea, sector formation was observed in seventeen isolates and no sector formation was observed in rest isolates. The radial growth of fifty isolates of P. grisea were ranged from 76.0 mm to 90.0 mm. Out of fifty isolates, group I included six isolates, group II and III included eleven and thirty three isolates, respectively. Parasexual recombination rarely causes genetic and phenotypic variation through hyphal anastomosis in India. Parasexual recombination is the principle cause in rice blast fungus and cause infection of resistant rice variety (IR-64) in India .

SummaryAsexual fungi include important pathogens of plants and other organisms, and their effective management requires understanding of their evolutionary dynamics. Genetic recombination is critical for species adaptability and could be achieved via heterokaryosis and the parasexual cycle in asexual fungi. Here, we investigate the extent and mechanisms of heterokaryosis in the asexual plant pathogen Verticillium dahliae.We used live-cell imaging and genetic complementation assays of tagged V. dahliae strains to analyze the extent of nonself vegetative fusion, heterokaryotic cell fate and nuclear behavior. An efficient CRISPR/Cas9-mediated system was developed to investigate the involvement of autophagy in heterokaryosis.Under starvation, nonself fusion of germinating spores occurs frequently regardless of the previously assessed vegetative compatibility of the partners. Supposedly “incompatible” fusions often establish viable heterokaryotic cells and mosaic mycelia, where nuclei can engage in fusion or transfer of genetic material. The molecular machinery of autophagy has a protective function against destruction of “incompatible” heterokaryons.Our results suggest an autophagy-mediated trade-off between parasexual interactions for genetic exchange and allorecognition systems possibly for mycelial protection from parasitic elements. Our study reveals unexpected capacity for heterokaryosis in V. dahliae and suggests, therefore, important roles of parasexuality in the evolution of asexual fungi.

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Arbuscular mycorrhizal fungi (AMF) are part of the most widespread fungal-plant symbiosis. They colonize at least 80% of plant species, promote plant growth and plant diversity. These fungi are multinucleated and contain either one or two haploid nuclear genotypes (monokaryon and dikaryon) identified by the alleles at a putative mating-type locus. This taxon has been considered as an ancient asexual scandal because of the lack of observable sexual structures. Despite identification of a putative mating-type locus and functional activation of genes related to mating when two isolates co-exist, it remains unknown if AMF take part in a mainly sexual life cycle.We used publicly available genome sequences to test if dikaryon genomes display signatures of sexual reproduction in the form of reciprocal recombination patterns, or if they display signatures of parasexual reproduction involving gene conversion.We used short-read and long-read sequence data to identify nucleus genotype-specific haplotypes within dikaryons and then compared them to orthologous gene sequences from related monokaryon isolates displaying the same putative MAT-types. We observed that these genotype-specific haplotypes display reciprocal recombination and not gene conversion.These results are consistent with a sexual origin of the dikaryon rather than a parasexual origin and provides an important step to understand the life cycle of these globally important symbiotic fungi.

The opportunistic fungal pathogen Candida albicans undergoes an unusual parasexual cycle wherein diploid cells mate to form tetraploid cells that can generate genetically diverse progeny via a nonmeiotic program of chromosome loss. The genetic diversity afforded by parasex impacts clinically relevant features including drug resistance and virulence, and yet the factors influencing genome instability in C. albicans are not well defined. To understand how environmental cues impact genome instability, we monitored ploidy change following tetraploid cell growth in a panel of different carbon sources. We found that growth in one carbon source, D-tagatose, led to high levels of genomic instability and chromosome loss in tetraploid cells. This sugar is a stereoisomer of L-sorbose which was previously shown to promote karyotypic changes in C. albicans. However, while expression of the SOU1 gene enabled utilization of L-sorbose, overexpression of this gene did not promote growth in D-tagatose, indicating differences in assimilation of the two sugars. In addition, genome sequencing of multiple progenies recovered from D-tagatose cultures revealed increased relative copy numbers of chromosome 4, suggestive of chromosome-level regulation of D-tagatose metabolism. Together, these studies identify a novel environmental cue that induces genome instability in C. albicans, and further implicate chromosomal changes in supporting metabolic adaptation in this species.

Rice blast disease is both the most explosive and potentially damaging disease of the world's rice (Oryza sativa) crop and a model system for research on the molecular mechanisms that fungi use to cause plant disease. The blast fungus, Magnaporthe oryzae, is highly evolved to sense when it is on a leaf surface; to develop a pressurized cell, the appressorium, to punch through the leaf cuticle; and then to hijack living rice cells to assist it in causing disease. Host specificity, determining which plants particular fungal strains can infect, is also an important topic for research. The blast fungus is a moving target, quickly overcoming rice resistance genes we deploy to control it, and recently emerging to cause devastating disease on an entirely new cereal crop, wheat. M. oryzae is highly adaptable, with multiple examples of genetic instability at certain gene loci and in certain genomic regions. Understanding the biology of the fungus in the field, and its potential for genetic and genome variability, is key to keep it from adapting to life in the research laboratory and losing relevance to the significant impact it has on global food security.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. In this study, we report that the spores of the important asexual plant-pathogenic fungusVerticillium dahliaeoften engage in cell fusion via Conidial Anastomosis Tubes (CATs). We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion inV. dahliaeas an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.

The concept of the biological species is often difficult to define with a reasonable degree of certainty, and this is perhaps especially true in fungi. The second class of incompatibility system found in fungi is heterogenically controlled. This incompatibility system acts primarily at the vegetative stage of the life cycle and as such has been variously referred to as vegetative, somatic, or heterokaryon incompatibility. An alternative explanation for the natural function of the vegetative incompatibility system has been suggested. Although the genetic analysis by conventional methods of vegetative incompatibility is possible within a sexual species such as A. nidulans, the use of protoplast fusion has greatly facilitated this analysis because it has permitted the more efficient parasexual methods to be used. Vegetative incompatibility in Aspergillus was first described in A. niger and subsequently in several other sexual and asexual species. An alternative explanation for the natural function of the vegetative incompatibility system has been suggested.

Candida albicans is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that C. albicans and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce C. albicans genome instability. Here, we investigated how C. albicans genetic background and ploidy state impact genome instability, both in vitro and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on C. albicans genetic background. Furthermore, we show that tetraploid C. albicans is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid C. albicans displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment., Candida albicans is an opportunistic fungal pathogen of humans that is typically diploid yet has a highly labile genome tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial for C. albicans to adapt to changing or stressful environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, in which tetraploids arise via diploid mating or stress-induced mitotic defects and undergo nonmeiotic ploidy reduction. However, it remains largely unknown how genetic background contributes to C. albicans genome instability in vitro or in the host environment. Here, we tested how genetic background, ploidy, and the host environment impacts C. albicans genome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied across C. albicans strain background and ploidy state. We then assessed if host-induced genomic changes resulted in fitness consequences on growth rate and nonlethal virulence phenotypes and found that many host-derived isolates significantly changed relative to their parental strain. Interestingly, diploid host-associated C. albicans predominantly decreased host reproductive fitness, whereas tetraploid host-associated C. albicans increased host reproductive fitness. Together, these results are important for understanding how host-induced genomic changes in C. albicans alter its relationship with the host. IMPORTANCE Candida albicans is an opportunistic fungal pathogen of humans. The ability to generate genetic variation is essential for adaptation and is a strategy that C. albicans and other fungal pathogens use to change their genome size. Stressful environments, including the host, induce C. albicans genome instability. Here, we investigated how C. albicans genetic background and ploidy state impact genome instability, both in vitro and in a host environment. We show that the host environment induces genome instability, but the magnitude depends on C. albicans genetic background. Furthermore, we show that tetraploid C. albicans is highly unstable in host environments and rapidly reduces in genome size. These reductions in genome size often resulted in reduced virulence. In contrast, diploid C. albicans displayed modest host-induced genome size changes, yet these frequently resulted in increased virulence. Such studies are essential for understanding how opportunistic pathogens respond and potentially adapt to the host environment.

Candida albicansis an opportunistic fungal pathogen of humans that is typically diploid yet, has a highly labile genome that is tolerant of large-scale perturbations including chromosomal aneuploidy and loss-of-heterozygosity events. The ability to rapidly generate genetic variation is crucial forC. albicansto adapt to changing or stress environments, like those encountered in the host. Genetic variation occurs via stress-induced mutagenesis or can be generated through its parasexual cycle, which includes mating between diploids or stress-induced mitotic defects to produce tetraploids and non-meiotic ploidy reduction. However, it remains largely unknown how genetic background contributes toC. albicansgenome instabilityin vitroorin vivo.Here, we tested how genetic background, ploidy and host environment impactC. albicansgenome stability. We found that host association induced both loss-of-heterozygosity events and genome size changes, regardless of genetic background or ploidy. However, the magnitude and types of genome changes varied acrossC. albicansstrains. We also assessed whether host-induced genomic changes resulted in any consequences on growth rate and virulence phenotypes and found that many host derived isolates had significant changes compared to their parental strains. Interestingly, host derivatives from diploidC. albicanspredominantly displayed increased virulence, whereas host derivatives from tetraploidC. albicanshad mostly reduced virulence. Together, these results are important for understanding how host-induced genomic changes inC. albicansalter the relationship between the host andC. albicans.

Certain Aspergillus species such as Aspergillus flavus and A. parasiticus are well known for the formation of sclerotia. These developmental structures are thought to act as survival structures during adverse environmental conditions but are also a prerequisite for sexual reproduction. We previously described an A. niger mutant (scl-2) which formed sclerotium-like structures, suggesting a possible first stage of sexual development in this species. Several lines of evidence presented in this study support the previous conclusion that the sclerotium-like structures of scl-2 are indeed sclerotia. These included the observations that: (i) safranin staining of the sclerotia-like structures produced by the scl-2 mutant showed the typical cellular structure of a sclerotium; (ii) metabolite analysis revealed specific production of indoloterpenes, which have previously been connected to sclerotium formation; (iii) formation of the sclerotium-like structures is dependent on a functional NADPH complex, as shown for other fungi forming sclerotia. The mutation in scl-2 responsible for sclerotium formation was identified using parasexual crossing and bulk segregant analysis followed by high throughput sequencing and subsequent complementation analysis. The scl-2 strain contains a mutation that introduces a stop codon in the putative DNA binding domain of a previously uncharacterized Zn(II)2Cys6 type transcription factor (An08g07710). Targeted deletion of this transcription factor (sclB) confirmed its role as a repressor of sclerotial formation and in the promotion of asexual reproduction in A. niger. Finally, a genome-wide transcriptomic comparison of RNA extracted from sclerotia versus mycelia revealed major differences in gene expression. Induction of genes related to indoloterpene synthesis was confirmed and also let to the identification of a gene cluster essential for the production of aurasperones during sclerotium formation. Expression analysis of genes encoding other secondary metabolites, cell wall related genes, transcription factors, and genes related to reproductive processes identified many interesting candidate genes to further understand the regulation and biosynthesis of sclerotia in A. niger. The newly identified SclB transcription factor acts as a repressor of sclerotium formation and manipulation of sclB may represent a first prerequisite step towards engineering A. niger strains capable of sexual reproduction. This will provide exciting opportunities for further strain improvement in relation to protein or metabolite production in A. niger.

Fungi and fungal-like organisms (oomycetes) that cause diseases in plants have impacted human communities for centuries and probably from the dawn of agriculture. In modern agriculture, there is a constant race between new strategies to manage fungal plant pathogens and their ability to adapt. An important component in this race is fungal genetic diversity. Mechanisms such as sexual and parasexual recombination that contribute to the creation of novel allele combinations in fungal plant pathogens are briefly discussed in the first part of this review. Advances in genomics have enabled the investigation of chromosomal aberrations of agriculturally important fungal isolates at the nucleotide level. Some of these cases are summarized in the second part of this review; it is claimed that the effect of chromosomal aberrations on pathogenicity should be studied mechanistically. More data on the effect of gene copy number variations on phenotypes that are relevant to agriculture are especially needed. Genome rearrangements through translocations have shaped the genome of fungal plant pathogens by creating lineage-specific chromosome territories encoding for genes participating in plant diseases. Pathogenicity chromosomes are unique cases of such lineage-specific genetic elements, interestingly these chromosomes can be transferred horizontally and thus transforming a non-pathogenic strain to a pathogenic one. The third part of this review describes our attempts to reveal mutators in fungal plant pathogens by identifying fungi that lack important DNA repair genes or respond to DNA damage in an unconventional way. We found that a group of fungal plant pathogens lack conserved genes that are needed for an important Holliday junction resolution pathway. In addition, in Fusarium oxysporum, the rate-limiting step in dNTP production is not induced under DNA replication stress. This is very different from organisms from bacteria to humans. It remains to be seen if these mechanisms promote genetic instability in fungal plant pathogens.

The initiation and progression of cancers reflect the underlying process of somatic evolution, in which the diversification of heritable phenotypes provides a substrate for natural selection, resulting in the outgrowth of the most fit subpopulations. Although somatic evolution can tap into multiple sources of diversification, it is assumed to lack access to (para)sexual recombination—a key diversification mechanism throughout all strata of life. On the basis of observations of spontaneous fusions involving cancer cells, the reported genetic instability of polypoid cells and the precedence of fusion-mediated parasexual recombination in fungi, we asked whether cell fusions between genetically distinct cancer cells could produce parasexual recombination. Using differentially labelled tumour cells, we found evidence of low-frequency, spontaneous cell fusions between carcinoma cells in multiple cell line models of breast cancer both in vitro and in vivo. While some hybrids remained polyploid, many displayed partial ploidy reduction, generating diverse progeny with heterogeneous inheritance of parental alleles, indicative of partial recombination. Hybrid cells also displayed elevated levels of phenotypic plasticity, which may further amplify the impact of cell fusions on the diversification of phenotypic traits. Using mathematical modelling, we demonstrated that the observed rates of spontaneous somatic cell fusions may enable populations of tumour cells to amplify clonal heterogeneity, thus facilitating the exploration of larger areas of the adaptive landscape (relative to strictly asexual populations), which may substantially accelerate a tumour’s ability to adapt to new selective pressures. Although tumours evolve in a Darwinian fashion, they do not undergo sexual reproduction. The authors show experimentally and using mathematical modelling that spontaneous fusion between cancer cells allows recombination between clones and the possibility of exploring larger areas of the adaptive landscape.

Initiation and progression of cancers reflect the underlying process of somatic evolution, which follows a Darwinian logic, i.e., diversification of heritable phenotypes provides a substrate for natural selection, resulting in the outgrowth of the most fit subpopulations. Although somatic evolution can tap into multiple sources of diversification, it is assumed to lack access to (para)sexual recombination – a key diversification mechanism throughout all strata of life. Based on observations of spontaneous fusions involving cancer cells, reported genetic instability of polypoid cells, and precedence of fusion-mediated parasexual recombination in fungi, we asked whether cell fusions could serve as a source of parasexual recombination in cancer cell populations. Using differentially labelled tumor cells, we found evidence of low-frequency, spontaneous cell fusions between carcinoma cells in multiple cell line models of breast cancer both in vitro and in vivo. While some hybrids remained polyploid, many displayed partial ploidy reduction, generating diverse progeny with heterogeneous inheritance of parental alleles, indicative of partial recombination. Hybrid cells also displayed elevated levels of phenotypic plasticity, which may further amplify the impact of cell fusions on the diversification of phenotypic traits. Using mathematical modeling, we demonstrated that the observed rates of spontaneous somatic cell fusions may enable populations of tumor cells to amplify clonal heterogeneity, thus facilitating the exploration of larger areas of the adaptive landscape, relative to strictly asexual populations, which may substantially accelerate a tumor’s ability to adapt to new selective pressures.