Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes.

Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called 'effectors'. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the 'pathogenicity' chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

EBR1 genomic expansion and its role in virulence of Fusarium species.

Genome sequencing of Fusarium oxysporum revealed that pathogenic forms of this fungus harbour supernumerary chromosomes with a wide variety of genes, many of which likely encode traits required for pathogenicity or niche specialization. Specific transcription factor gene families are expanded on these chromosomes including the EBR1 family (Enhanced Branching). The significance of the EBR1 family expansion on supernumerary chromosomes and whether EBR1 paralogues are functional is currently unknown. EBR1 is found as a single copy in F.graminearum and other fungi but as multiple paralogues in pathogenic F.oxysporum strains. These paralogues exhibit sequence and copy number variation among different host-specific strains and even between more closely related strains. Relative expression of the EBR1 paralogues depends on growth conditions and on the presence of the single EBR1 gene in the core genome. Deletion of EBR1 in the core genome in different F.oxysporum strains resulted in impaired growth, reduced pathogenicity and slightly reduced biocontrol capacities. To identify genes regulated by EBR1, the transcriptomes of wild-type and Δebr1 strains were compared for both F.oxysporum and F.graminearum. These studies showed that in both species, EBR1 regulates genes involved in general metabolism as well as virulence.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates.

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.

Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells.

Fusarium oxysporum is an asexual, soil inhabiting fungus that comprises many different formae speciales, each pathogenic towards a different host plant. In absence of a suitable host all F. oxysporum isolates appear to have a very similar lifestyle, feeding on plant debris and colonizing the rhizosphere of living plants. Upon infection F. oxysporum switches from a saprophytic to an infectious lifestyle, which probably includes the reprogramming of gene expression. In this work we show that the expression of the known effector gene SIX1 of F. oxysporum f. sp. lycopersici is strongly upregulated during colonization of the host plant. Using GFP (green fluorescent protein) as reporter, we show that induction of SIX1 expression starts immediately upon penetration of the root cortex. Induction requires living plant cells, but is not host specific and does not depend on morphological features of roots, since plant cells in culture can also induce SIX1 expression. Taken together, F. oxysporum seems to be able to distinguish between living and dead plant material, preventing unnecessary switches from a saprophytic to an infectious lifestyle.

Virulence genes and the evolution of host specificity in plant-pathogenic fungi.

In the fungal kingdom, the ability to cause disease in plants appears to have arisen multiple times during evolution. In many cases, the ability to infect particular plant species depends on specific genes that distinguish virulent fungi from their sometimes closely related nonvirulent relatives. These genes encode host-determining "virulence factors," including small, secreted proteins and enzymes involved in the synthesis of toxins. These virulence factors typically are involved in evolutionary arms races between plants and pathogens. We briefly summarize current knowledge of these virulence factors from several fungal species in terms of function, phylogenetic distribution, sequence variation, and genomic location. Second, we address some issues that are relevant to the evolution of virulence in fungi toward plants; in particular, horizontal gene transfer and the genomic organization of virulence genes.

Adaptation to the Host Environment by Plant-Pathogenic Fungi.

Many fungi can live both saprophytically and as endophyte or pathogen inside a living plant. In both environments, complex organic polymers are used as sources of nutrients. Propagation inside a living host also requires the ability to respond to immune responses of the host. We review current knowledge of how plant-pathogenic fungi do this. First, we look at how fungi change their global gene expression upon recognition of the host environment, leading to secretion of effectors, enzymes, and secondary metabolites; changes in metabolism; and defense against toxic compounds. Second, we look at what is known about the various cues that enable fungi to sense the presence of living plant cells. Finally, we review literature on transcription factors that participate in gene expression in planta or are suspected to be involved in that process because they are required for the ability to cause disease.

Horizontal transfer of supernumerary chromosomes in fungi.

Several species of filamentous fungi contain so-called dispensable or supernumerary chromosomes. These chromosomes are dispensable for the fungus to survive, but may carry genes required for specialized functions, such as infection of a host plant. It has been shown that at least some dispensable chromosomes are able to transfer horizontally (i.e., in the absence of a sexual cycle) from one fungal strain to another. In this paper, we describe a method by which this can be shown. Horizontal chromosome transfer (HCT) occurs during co-incubation of two strains. To document the actual occurrence of HCT, it is necessary to select for HCT progeny. This is accomplished by transforming two different drug-resistance genes into the two parent strains before their co-incubation. In one of the strains (the "donor"), a drug-resistance gene should be integrated in a chromosome of which the propensity for HCT is under investigation. In the "tester" or "recipient" strain, another drug-resistance gene should be integrated somewhere in the core genome. In this way, after co-incubation, HCT progeny can be selected on plates containing both drugs. HCT can be initiated with equal amounts of asexual spores of both strains, plated on regular growth medium for the particular fungus, followed by incubation until new asexual spores are formed. The new asexual spores are then harvested and plated on plates containing both drugs. Double drug-resistant colonies that appear should carry at least one chromosome from each parental strain. Finally, double drug-resistant strains need to be analysed to assess whether HCT has actually occurred. This can be done by various genome mapping methods, like CHEF-gels, AFLP, RFLP, PCR markers, optical maps, or even complete genome sequencing.

Drifter, a novel, low copy hAT-like transposon in Fusarium oxysporum is activated during starvation.

The facultative pathogenic fungus Fusarium oxysporum is known to harbour many different transposable and/or repetitive elements. We have identified Drifter, a novel DNA transposon of the hAT family in F. oxysporum. It was found adjoining SIX1-H, a truncated homolog of the SIX1 avirulence gene in F. oxysporum f. sp. lycopersici. Absence of a target site duplication as well as the 5' part of SIX1-H suggests that transposition of Drifter into the ancestor of SIX1-H was followed by loss of a chromosomal segment through recombination between Drifters. F. oxysporum isolates belonging to various formae speciales harbour between 0 and 5 full-length copies of Drifter and/or one or more copies with an internal deletion. Transcription of Drifter is activated during starvation for carbon or nitrogen.

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato.

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.