Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype ofAlternaria alternata
Authors: Wang Mingshuang , Fu Huilan , Shen Xing-Xing , Ruan Ruoxin , Pun Nicholas , Xu Jianping , Li Hongye , Rokas Antonis 
Published: November 2017

The tangerine pathotype of the ascomycete fungusAlternaria alternatais 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 ofA. 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 theA. alternataZ7 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 H2O2treatment 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 genusAlternariaand 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 fromColletotrichum(Sordariomycetes) and 5 were likely transferred as a physically linked cluster of genes fromCryptococcus(Basidiomycota) orPenicillium(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 inAlternaria.

Author Summary

Many 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 ofAlternaria alternataZ7 strain. We found that most of the Z7 CDC proteins are highly conserved within the genusAlternariabut 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 fromColletotrichum, and a cluster of 5 physically linked genes likely transferred fromCryptococcus(Basidiomycota) orPenicillium(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 thatAlternariaCDCs 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.