MOLECULAR PLANT PATHOLOGY (2019) 20(10), 1425–1438 DOI: 10.1111/mpp.12848 Genomic features and evolution of the conditionally dispensable chromosome in the tangerine pathotype of Alternaria alternata MINGSHUANG WANG 1,2,3,†, HUILAN FU1,†, XING-XING SHEN2, RUOXIN RUAN1,4, ANTONIS ROKAS2 AND HONGYE LI1,* 1 Key Lab of Molecular Biology of Crop Pathogens and Insects, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China 2 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA 3 College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China 4 Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China SUMMARY INTRODUCTION The tangerine pathotype of the ascomycete fungus Alternaria Alternaria alternata fungi can be ubiquitously found in soil, var- alternata is the causal agent of citrus brown spot, which can ious plants, and decaying plant debris (Thomma, 2003). Some result in significant losses of both yield and marketability for A. alternata strains can cause plant diseases and result in se- tangerines worldwide. A conditionally dispensable chromosome vere crop losses worldwide. Because these strains can often be (CDC), which harbours the host-selective ACT toxin gene clus- morphologically very similar but exhibit substantial pathological ter, is required for tangerine pathogenicity of A. alternata. To differences, they have been defined as pathotypes of A. alter- understand the genetic makeup and evolution of the tangerine nata (Tsuge et al., 2013). At least seven pathogenic A. alternata pathotype CDC, we isolated and sequenced the CDCs of the pathotypes, each producing a unique host-selective toxin (HST) A. alternata Z7 strain and analysed the function and evolution essential to pathogenicity, have been recognized to cause dis- of their genes. The A. alternata Z7 strain has two CDCs (~1.1 eases in Japanese pear, strawberry, tangerine, apple, tomato, and ~0.8 Mb, respectively), and the longer Z7 CDC contains rough lemon and tobacco (Tsuge et al., 2013). Generally, genes all but one contig of the shorter one. Z7 CDCs contain 254 required for HST biosynthesis in A. alternata are clustered on rel- predicted protein-coding genes, which are enriched in func- atively small chromosomes that are 1.0–2.0 Mb in size (Tsuge tional categories associated with ‘metabolic process’ (55 genes, et al., 2013). These accessory or conditionally dispensable chro- P = 0.037). Relatively few of the CDC genes can be classified mosomes (CDCs) are highly variable among species, are gener- as carbohydrate-active enzymes (CAZymes) (4) and transporters ally not required for growth and reproduction on artificial media, (19) and none as kinases. Evolutionary analysis of the 254 CDC but are required for A. alternata pathogenicity (Hatta et al., proteins showed that their evolutionary conservation tends to be 2002; Johnson et al., 2001). restricted within the genus Alternaria and that the CDC genes The importance of CDCs conferring pathogenicity to fruits evolve faster than genes in the essential chromosomes, likely has been demonstrated by the construction of A. alternata hy- due to fewer selective constraints. Interestingly, phylogenetic brids in laboratory conditions. More specifically, two distinct lab- analysis suggested that four of the 25 genes responsible for the oratory hybrids were constructed from tomato and strawberry ACT toxin production were likely transferred from Colletotrichum pathotypes and separately for apple and tomato pathotypes. (Sordariomycetes). Functional experiments showed that two of The resulting hybrids harboured two CDCs from their parents, them are essential for the virulence of the tangerine pathotype of allowing for the production of HSTs that caused diseases in both A. alternata. These results provide new insights into the function parental plant species (Akagi et al., 2009b; Akamatsu et al., and evolution of CDC genes in Alternaria. 2001). Furthermore, these studies support the hypothesis that CDCs can be transmitted between different strains thereby fa- Keywords: accessory chromosome, Dothideomycetes, cilitating the spread and evolutionary diversification of fungal evolutionary origin, horizontal gene transfer, karyotype, phytopathogens. pathogenicity. Besides A. alternata, many other fungal phytopathogens have CDCs in their genomes, including Fusarium oxyspo- *Correspondence: Email: [email protected] rum, Nectria haematococca, Zymoseptoria tritici (previously †These authors contributed equally to this work. known as Mycosphaerella graminicola and Septoria tritici) and © 2019 THE AUTHORS. MOLECULAR PLANT PATHOLOGY PUBLISHED BY BRITISH SOCIETY FOR PLANT PATHOLOGY AND JOHN WILEY & SONS LTD This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 1425 1426 M. WANG et al. Colletotrichum gloeosporioides (Coleman et al., 2009; Ma et al., genes was restricted to the genus Alternaria and that four CDC 2010; Stukenbrock et al., 2010). Because CDCs are typically genes belonging to the ACT toxin gene cluster likely originated found in some, but not all, strains, they have been proposed to via horizontal gene transfer (HGT) events from other fungi. The have different evolutionary origins than the essential chromo- recombination mutants of two HGT genes showed a dramatic somes (Covert, 1998; Tsuge et al., 2013), e.g. through horizontal reduction in virulence to citrus leaves. These results suggest transfer from distantly related species (Covert, 1998; Hatta et al., that Alternaria CDCs exhibit chromosomal polymorphisms, 2002). The possibility of horizontal transfer of CDCs between that CDC genes are involved in processes associated with me- species has been demonstrated in several studies. For example, tabolism, that they are rapidly evolving and that the charac- a 2 Mb chromosome was transferred between two different bio- teristics associated with their virulence sometimes originate types of C. gloeosporioides during vegetative co-cultivation in via HGT. the laboratory (He et al., 1998). Importantly, CDC acquisition can facilitate the transition from the non-pathogenic to pathogenic RESULTS phenotype in the recipient organism. For example, co-incubation of non-pathogenic and pathogenic genotypes of F. oxysporum Identification and sequencing of the CDCs revealed that the transfer of a CDC from the pathogenic (donor) The tangerine pathotype strain of A. alternata from Florida con- to non-pathogenic genotype (recipient) enabled the recipient to tains one CDC (Miyamoto et al., 2009), which led us to reason become pathogenic to tomatoes (Ma et al., 2010). that the A. alternata Z7 strain would also contain one CDC. To Previously, the ~1.0 Mb CDC of the tomato pathotype of A. al- evaluate the genomic size of the CDC in A. alternata Z7, we per- ternata was identified and characterized (Hu et al., 2012). Genes formed pulsed-field gel electrophoresis using Z7 protoplast cells. in that CDC are abundant in the categories of ‘metabolic pro- Unexpectedly, we found that the strain Z7 possesses two CDCs, cess’ and ‘biosynthetic process’, and include 36 polyketide and one that is ~1.1 Mb in size and another that is ~0.8 Mb (Fig. 1). non-ribosomal peptide synthetase domain-containing genes. The electrophoretic karyotype of A. alternata Z7 is different from Furthermore, the G+C content of third codon positions, codon that of the tangerine pathotype strains isolated from Florida, usage bias and repeat region load in the CDC are different from USA, which has only one ~1.9–2.0 Mb CDC (Miyamoto et al., those in the essential chromosomes (Hu et al., 2012), raising the 2009). These results suggest that Alternaria strains within the hypothesis that the A. arborescens CDC was acquired through same pathotype exhibit variability in both the number and size horizontal transfer from an unrelated fungus (Hu et al., 2012). of their CDCs. The tangerine pathotype of A. alternata, which can cause To obtain the genomic sequence of the A. alternata Z7 CDCs, citrus brown spot on tangerines and tangerine hybrids, was we isolated the corresponding CDC bands from the pulsed-field demonstrated to harbour an additional chromosome of about 1.9–2.0 Mb by pulsed-field gel electrophoresis studies (Miyamoto et al., 2008, 2009). Seven genes, ACTTR, ACTT2, ACTT3, ACTT5, ACTT6, ACTTS2 and ACTTS3, were located on this chromosome and found to be required for the biosynthe- sis of the ACT toxin, a unique HST produced by the tanger- ine pathotype (Tsuge et al., 2013). However, relatively little is known about the content, potential biological functions and evolution of the other genes in the CDC of the tangerine pathotype of A. alternata. To address this question, we isolated and sequenced the CDCs of the tangerine pathotype strain Z7 of A. alternata iso- lated from Zhejiang, China (Huang et al., 2015). We found that the Z7 strain contains two different CDCs, 1.1 and 0.8 Mb in size; in contrast, another strain of the same pathotype from Florida was described to have one CDC (Miyamoto et al., 2009). Interestingly, the smaller CDC is nearly identical in se- quence to part of the larger CDC in the Z7 strain. Examination of the functional annotation of all 254 genes in the Z7 CDCs showed that 55 out of the 254 CDC genes are enriched in func- Fig. 1 Electrophoretic karyotype of the Alternaria alternata Z7 tions associated