A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

A pair of effectors encoded on a conditionally dispensable chromosome of Fusarium oxysporum suppress host-specific immunity

Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute significantly to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis and cabbage. Here we show that Focn has multiple CD chromosomes involved in virulence but also in vegetative growth, which is an atypical feature of CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.

Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae – a pathogen of the Brassicaceae family

Background Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. Results We also sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea using Nanopore MinION sequencing for additional comparative analyses within the Alternaria genus. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae, a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae-specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae. Conclusion The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae.

A Sequence Resource of Autosomes and Additional Chromosomes in the Peach Pathotype of Alternaria alternata

Alternaria alternata is a generally saprophytic fungus. Its genome consists of 10 autosomes, while some strains have one or two additional chromosomes, called a conditionally dispensable chromosome (CDC). A CDC is not required for reproduction but confers host-specific pathogenicity. We sequenced the genome of the peach pathotype of A. alternata using Nanopore and assembled it into 20 sequences. The 10 largest sequences corresponded to 10 gapless sequences of A. solani autosomes, and 1 sequence was a mitochondrial genome. Nine other sequences may be derived from CDCs because of lack of similarity with autosomes of other Alternaria spp. The sequence information could provide novel insights into genomes of Alternaria spp. and on the biosynthesis of a novel host-specific toxin in the peach pathotype of A. alternata.

Comparative genomics of Alternaria species provides insights into the pathogenic lifestyle of Alternaria brassicae – a pathogen of the Brassicaceae family

Alternaria brassicae, a necrotrophic pathogen, causes Alternaria Leaf Spot, one of the economically important diseases of Brassica crops. Many other Alternaria spp. such as A. brassicicola and A. alternata are known to cause secondary infections in the A. brassicae-infected Brassicas. The genome architecture, pathogenicity factors, and determinants of host-specificity of A. brassicae are unknown. In this study, we annotated and characterised the recently announced genome assembly of A. brassicae and compared it with other Alternaria spp. to gain insights into its pathogenic lifestyle. Additionally, we sequenced the genomes of two A. alternata isolates that were co-infecting B. juncea. Genome alignments within the Alternaria spp. revealed high levels of synteny between most chromosomes with some intrachromosomal rearrangements. We show for the first time that the genome of A. brassicae, a large-spored Alternaria species, contains a dispensable chromosome. We identified 460 A. brassicae-specific genes, which included many secreted proteins and effectors. Furthermore, we have identified the gene clusters responsible for the production of Destruxin-B, a known pathogenicity factor of A. brassicae. The study provides a perspective into the unique and shared repertoire of genes within the Alternaria genus and identifies genes that could be contributing to the pathogenic lifestyle of A. brassicae.

Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype of Alternaria alternata

The tangerine pathotype of the ascomycete fungus Alternaria alternata is the causal agent of citrus brown spot, which can result in significant losses of both yield and marketability for tangerines and tangerine hybrids worldwide. A conditionally dispensable chromosome (CDC), which harbors the host-selective ACT toxin gene cluster, is required for tangerine pathogenicity of A. alternata. To understand the genetic makeup and evolution of the tangerine pathotype CDC, we analyzed the function and evolution of the CDC genes present in the A. alternata Z7 strain. The 1.84Mb long CDC contains 512 predicted protein-coding genes, which are enriched in functional categories associated with ‘metabolic process’ (132 genes, p-value = 0.00192) including ‘oxidation-reduction process’ (48 genes, p-value = 0.00021) and ‘lipid metabolic process’ (11 genes, p-value = 0.04591). Relatively few of the CDC genes can be classified as CAZymes (13), kinases (3) and transporters (20). Differential transcriptome analysis of H2O2 treatment and control conditions revealed that 29 CDC genes were significantly up-regulated and 14 were significantly down-regulated, suggesting that CDC genes may play a role in coping with oxidative stress. Evolutionary analysis of the 512 CDC proteins showed that their evolutionary conservation tends to be restricted within the genus Alternaria and that the CDC genes evolve faster than genes in the essential chromosomes. Interestingly, phylogenetic analysis suggested that the genes of 13 enzymes and one sugar transporter residing in the CDC were likely horizontally transferred from distantly related species. Among these, one carboxylesterase gene was transferred from bacteria but functionally knocking out this gene revealed no obvious biological role. Another 4 genes might have been transferred from Colletotrichum (Sordariomycetes) and 5 were likely transferred as a physically linked cluster of genes from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). Functionally knocking out the 5-gene cluster resulted in an 80% decrease in asexual spore production in the deletion mutant. These results provide new insights into the function and evolution of CDC genes in Alternaria.Author SummaryMany fungal phytopathogens harbor conditionally dispensable chromosomes (CDCs). CDCs are variable in size, contain many genes involved in virulence, but their evolution remains obscure. In this study, we investigated the origin of the CDC present in the tangerine pathotype of Alternaria alternata Z7 strain. We found that most of the Z7 CDC proteins are highly conserved within the genus Alternaria but poorly conserved outside the genus. We also discovered that a small number of genes originated via horizontal gene transfer (HGT) from distantly related fungi and bacteria. These horizontally transferred genes include a carboxylesterase gene that was likely acquired from bacteria, a cluster of 4 physically linked genes likely transferred from Colletotrichum, and a cluster of 5 physically linked genes likely transferred from Cryptococcus (Basidiomycota) or Penicillium (Eurotiomycetes). To gain insight into the functions of these transferred genes, we knocked out the bacterial carboxylesterase and the 5-gene cluster. Whereas the carboxylesterase deletion mutant showed no obvious phenotype, the 5-gene cluster mutant showed a dramatically reduced production of asexual spores (conidia). The results of our study suggest that Alternaria CDCs are largely comprised from rapidly evolving native genes; although only a few genes were acquired via horizontal gene transfer, some of them appear to be involved in functions critical to the phytopathogenic lifestyle.