Genome-wide identification, phylogenetic analysis, and expression profiling of ATP-binding cassette transporters in oomycetes

doi:10.21203/rs.3.rs-1957985/v1

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Genome-wide identification, phylogenetic analysis, and expression profiling of ATP-binding cassette transporters in oomycetes

https://doi.org/10.21203/rs.3.rs-1957985/v1

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Background： ABC transporters are a special family of membrane proteins and fulfill a multitude of functions. In plants, a few research have found ABC transporters acting as virulence factors in oomycete-plant interactions. However, previous studies have only focused on phylogenetic analysis of ABC transporters in particular species, and a comprehensive analysis has not been carried out. In this study, we examined 21 species genomes of oomycetes for phylogenetic and functional analysis of ABC transporters. Aim to find potential virulence factors in the interaction of plants and oomycetes.

Result：We identified 29~151 ABC transporter proteins in 21 species of oomycetes, and 965 were filtered in a phylogenetic analysis. The number of ABC transporters in biotrophic oomycetes was significantly lower than in hemi-biotrophic and necrotrophic oomycetes, and the number in Phytophthorawas the largest. Our analysis demonstrated that most oomycete ABC transporters are highly conserved, while others show remarkable group-specific diversification. The ABCA group III and ABCB half-size II proteins clustered with bacteria, appearing to be derived from horizontal transfer events from bacterial genomes. The ABCG subfamily was the most expanded group in oomycetes. The number of ABCD subfamily members was the smallest and was highly conserved in oomycetes. The structural characteristics of ABC proteins indicate diverse structural types in each of the ABC subfamilies. A heatmap was used to analyze the expression of ABC transporters. ABC transporters were expressed in middle and late stages, which represent biotrophic and necrotrophic phases, with the exception of some ABCB transporters being highly expressed in early stage. This suggests that ABC proteins expressed in middle and late stages may participate in host disease and cause host cell death.

Conclusions: In this study, 965 proteinswere filtered in a phylogenetic analysis from 21 species of oomycete representing three different lifestyles. Meanwhile, we also analysis of oomycete ABC proteins provides insight into the diversity of the five subfamilies among the main oomycete lineages. This study presents the first comprehensive overview of the ABC transporter family, and provides an important platform for in-depth analysis of oomycete-plant interactions and a database for identifying pathogenic factors in oomycetes.

ABC transporters

Pathogenicity

Oomycete

Biotrophic

Necrotrophic

Expression

ATP-binding cassette (ABC) transporters are a special family of membrane proteins and one of the largest protein families, which are characterized by homologous ATP-binding, and large, multi-spanning transmembrane domains [1]. These proteins operate in all living cells of Archaea, Eubacteria, and Eukarya, and fulfill a remarkable variety of cellular functions [2.3]. The ABC transporter superfamily is involved in tolerance to a wide diversity of cytotoxic agents in both prokaryotes and eukaryotes [4]. This family usually plays a role in ATP-dependent transport of diverse substrates across biological membranes, including simple ions (polar, amphipathic and hydrophobic organic molecules), peptides, complex lipids and small proteins [5]. Consequently, ABC transporters are related to a wide array of developmental processes [6–11]. The basic structure of ABC full-length transporters has two nucleotide-binding domains (NBDs) that bind to and hydrolyze ATP and two transmembrane domains that each contain six transmembrane helices. The ABC half-size transporters only have one NBD and one transmembrane domain, the most common of which are ABCB half-size transporters. The NBD has five highly conserved motifs, including Walker A and B, the ABC signature motif, the H loop, and the Q loop [12]. The ABC signature (LSGGQ) motif links the Walker A and Walker B motifs. Distinct from other subfamilies, the characteristic feature of ABCG transporters is their topology, in which NBD precedes the transmembrane domain [7].

In eukaryotes, ABC proteins are classified in eight subfamilies (A–H) [5]. In ABCA to ABCD subfamilies, the characteristic feature is their forward TMD-NBD domain, while the ABCG subfamily is characterized by a reversed NBD-TMD organization [13]. ABCE and ABCF subfamilies consist of only two NBDs. They have other functions except transport which not associating with membrane [13.14]. The most prominent members of the ABC subfamily are the multi-drug resistance proteins (MDR, P-glycoprotein, and ABCB), multi-drug resistance-related proteins (MRPs, ABCC), and pleiotropic drug resistance (PDR, ABCG) [15.16]. ABC transporters are driven by ATP hydrolysis and can act as importers, exporters, receptors, and channels [17]. They are localized in most membranes of the cell such as the plasma membrane, the tonoplast, chloroplasts, mitochondria, and peroxisomes, and they fulfill a multitude of functions [13].

In most organisms, ABC transporters are among the largest membrane proteins which have widely divergent functions, especially in drug resistance. In humans, ABC transporters are causative factors in many clinically relevant pathologies, such as ABCC7 a chloride transporter result in cystic fibrosis; in plants, these transporters have important functions in pathogen response and numerous aspects of development, and these can affect global food security; in microbes, these transporters are also very important in virulence and drug resistance [5]. For instance, in yeast, The ATP-binding cassette superfamily proteins Pdr5p and Snq2p are implicated in multidrug resistance, and both of them are multidrug transporters with overlapping substrate specificities [18]. In fungi, the virulence factor BcatrB has been identified from Botrytis cinerea, which is an ABC transporter that plays a role in the efflux of fungi toxic compounds [19]. Although a few research have found ABC transporters acting as virulence factors in oomycete-plant interactions, there have been more studies showing that these transporters are highly expressed during the interaction; for example, in Aphanomyces euteiches, Phytophthora sojae, and Pythium ultimum [20.21]. These have been suggested to help eliminate phytoalexins liberated from lysing cells [21]. As more and more oomycete genomes and transcriptomes are sequenced, we find an astonishing diversity of ABC transporter proteins, providing a unique opportunity for comparative analysis within this group of organisms. Previous studies have only focused on phylogenetic or functional analysis of ABC transporters in particular species, and a comprehensive analysis has not been carried out. In this study, we examined 21 species genomes of oomycetes for phylogenetic analysis of ABC transporters. Our aim was to find potential virulence factors in the interaction of plants and oomycetes.

Identification of oomycete ABC transporters

We identified 29 ~ 151 ABC transporter proteins in 21 species of oomycetes (Table 1). The number of ABC transporters in biotrophic oomycetes (Bremia lactuae, Hyloperonospora arabidopsidis, Peronospora, Plasmopara halstedii included in Peronospora and Albugo) was significantly lower than in hemi-biotrophic and necrotrophic oomycetes, and the number in Phytophthora was the largest. This phenomenon of unbalanced distribution in oomycetes may be caused by their different lifestyles. Hemi-biotrophic Phytophthora contain biotrophic and necrotrophic phases and need more transporters for a broad range of special substrates. The ABCG subfamily was the most expanded group in oomycetes and accounted for over 50% in many species. In contrast, the number of ABCD subfamily members was the smallest and was highly conserved in oomycetes. The structural characteristics of ABC proteins indicate diverse structural types in each of the ABC subfamilies (Fig. 1). The ABCD subfamily only had one transmembrane domain. To further understand the evolutionary placement of oomycete ABC transporters, in-depth phylogenetic analyses were conducted for each subfamily. The results of these analyses are shown and discussed in the following sections.

Table 1

Numbers of proteins in the ABC families of selected oomycete genomes.
	Species Name	Total	ABCA	ABCB	ABCC	ABCD	ABCE	ABCF	ABCG	ABCI
Obligate biotroph	Albugo candida	54	4	9	28	2	1	1	11	0
Obligate biotroph	Albugo laibachii	73	24	7	18	0	1	2	21	0
Necrotroph	Aphanomyces astaci	45	6	17	7	1	1	0	12	2
Necrotroph	Aphanomyces invadans	47	8	18	6	1	1	0	12	2
Obligate biotroph	Bremia lactucae	43	3	22	4	1	0	1	12	1
	Hyloperonospora arabidopsidis	28	3	5	4	1	1	1	14	0
	Peronospora effusa Race 13	24	2	8	1	1	1	1	11	0
	Peronospora effusa Race 14	19	2	5	1	1	1	1	9	0
	Peronospora tabacina J2	38	1	12	2	0	2	1	18	2
	Peronospora tabacina S26	29	3	11	3	1	2	1	8	1
	Plasmopara halstedii	25	4	8	1	1	1	1	9	1
Hemibiotroph	Phytophthora infestans	85	14	15	12	1	1	1	41	1
	Phytophthora sojae	107	11	21	18	1	1	1	54	1
	Phytophthora ramorum	92	14	18	8	1	1	1	49	1
Necrotroph	Pythium vexans	72	12	16	7	1	1	1	34	1
	Pythium aphanidermatum	62	8	19	11	1	1	1	21	1
	Pythium arrhenomanes	42	8	14	8	1	1	0	10	1
	Pythium irregular	67	13	14	11	1	1	1	26	1
	Pythium iwayamai	53	6	12	14	0	1	1	19	0
	Pythium ultimum	111	14	27	27	1	1	1	40	1
	Saprolegnia diclina vs20	78	12	22	19	1	1	0	23	1
	Saprolegnia parasitica CBS 223.65	89	15	25	23	1	1	0	24	1

Phylogenetic analysis of ABCA subfamily

We filtered a total of 117 full-length ABCA transporter proteins in 21 oomycete species containing over nine transmembrane helices. The phylogenetic tree indicates that the 117 ABCA proteins can be clustered in three different groups (I, II and III) (Fig. 2a). ABCA proteins of Phytophthora, Peronospora, Pythium, Albugo, and Saprolegnia were classified in three groups and the genome sequences showed that they were well conserved in oomycete species, though they were absented in a few species. For example, ABCA group III was absent in oomycete species. There were no ABCA proteins in Peronospora from group II, and Peronospora effusa R14 and Pythium arrhenomanes also lacked group III. This revealed an uneven distribution of ABCA group II and III proteins among oomycetes. To investigate the role of the three groups of ABCA transporters in oomycete species, we built a phylogenetic tree with Ph. infestans (as representative of oomycetes), Homo sapiens, plants, fungi, and bacteria (Fig. 2b). We found that group I and II clustered in a branch, and that group III was separate. The phylogenetic relationships of group I and II were closer to ABCA1 of H. sapiens and plants. The phylogenetic relationship of group III (XP_002909428.1) was closer to bacteria.

Phylogenetic analysis of multi-drug resistance (MDR)/ABCB subfamily

We filtered 155 ABCB full-length and 147 ABCB half-size transporters in oomycete species. To analyze the evolutionary relationship, we built phylogenetic trees of ABCB full-length and half-size proteins (Fig. 3a and Fig. 3b). ABCB full-length proteins clustered in four groups (I, II, III and IV) (Fig. 3a), and all those in Aphanomyces clustered in a separate branch. ABCB half-size transporters clustered in six groups and show more diverse evolution (I, II, III, IV, V and VI) (Fig. 3b). Group I only included ABCB half-size proteins of Saprolegnia and Aphanomyces. To further investigate the evolutionary relationships between the ABCB proteins of oomycetes and exogenous species, we built a phylogenetic tree of oomycetes (Ph. infestans and Ph. sojae as representatives), H. sapiens, plants, fungi, and bacteria. ABCB full-length group IV was separated from groups II and III, and clustered with the MAD20250.1 protein of Planctomycetaceae bacterium, and the relationship of group IV proteins is closer to bacteria (Fig. 4a). In the ABC-B half-size proteins (Fig. 4b), we found homologous ABCB6, ABCB7, ABCB10, ABCB25, and Atm proteins in H. sapiens, plants, and fungi, and they are all mitochondrial proteins. ABCB half-size group III/IV is closer to ABCB10, group II is closer only to ABCB of bacteria, and group V and VI are closer to ABCB7/ABCB25/Atm.

Phylogenetic analysis of MRP/ABCC subfamily

There are 218 ABCC proteins of 21 oomycete species that we filtered to build a phylogenetic tree. The 218 ABCC proteins clustered in six groups, including two big branches (I and V) and four small branches (II, III, IV and VI) (Fig. 5a). There are three orthologous groups in group I and two orthologous groups in group V. This revealed an extremely uneven distribution of ABCC evolution in oomycetes. Interestingly, there were no downy mildew ABCC proteins in ABCC group I and only one protein (CEG35276, Plasmopara halstedii) in ABCC group V. Meanwhile, ABCC proteins of Aphanomyces and Saprolegnia were clustered within sub-branches of the two large groups. Also, there were no ABCC proteins in Albugo from group I and V. There are only partial species in groups III, IV, and VI. ABCC proteins appear to be uneven in their evolutionary distribution and species diversity. In the phylogenetic tree of oomycetes, H. sapiens, plants, fungi, and bacteria, oomycete ABCC proteins clustered independently, except for groups II and IV (Fig. 5b). ABCC group II clustered with fungi YCF1/MdlB, plant MRP1-like and H. sapiens MRP1 proteins. ABCC group IV clustered with plant ABCC13 and human MRP7 with a bootstrap value of 93%.

Phylogenetic analysis of ABCD subfamily

All of the ABCD proteins identified in oomycetes were half-size and clustered together. Different from other oomycete species, Ph. sojae, Ph. ramorum, and Aphanomyces astaci contain two ABCD proteins, and Peronospora tabacina J2, Hyloperonospora arabidopsidis, Aphanomyces invadans, and Albugo candida have none (Fig. 6). The two ABCD prtoeins of Aphanomyces astaci were duplicated. ABCD proteins are not present in every oomycete species. Interestingly, we found RxLR and RxLR-like residues in ABCD proteins (Fig. 7). To further investigate the evolutionary relationships between the ABCD proteins of oomycetes and exogenous species, we built a phylogenetic tree of oomycetes (Ph. infestans and Ph. sojae as representatives), H. sapiens, plants, fungi, and bacteria. ABCD proteins of oomycetes did not cluster with any proteins from fungi or bacteria.

Phylogenetic analysis of ABCG subfamily

We filtered 308 full-length ABCG prtoeins in 21 oomycete species. To analyze their evolutionary relationship, we built a phylogenetic tree of ABCG full-length proteins. The 308 ABCG proteins were clustered in seven different groups (I, II, III, IV, V, VI and VII) (Fig. 8a). Although groups II, III, and VI have several small paralogous clades that did not cluster together, homologous sequence analysis of the small clades using the Orthofinder program showed that they belong to their closer, bigger clades. The small clades were separated, including Saprolegnia and Aphanomyce in group II, Albugo and Pythium in group III, and Pythium in group VI. Phytophthora and Peronospora all clustered together. Only Phytophthora and Pythium were involved in group IV. To obtain better insight into the role of the ABCG subfamily in oomycetes, a phylogenetic tree was generated with other proteins available from H. sapiens, plants, fungi, and bacteria (Fig. 8b). The phylogenetic tree showed that ABCG proteins are divided into three parts. Group I and group VII were separated from ABCG groups II/III/IV/V/VI in oomycetes. Group II/III/IV/V/VI and group I proteins clustered with ABC2 and PDR1 of plants. ABC2 is the first ABC transporter found in Magnaporthe grisea. To investigate the functions of all ABCG group VII members in oomycetes, we checked the functional sites of conserved domains in oomycetes, plant PDR1, and human ABCG1. We found that all group VII proteins contain two BND while other groups only have one.

Overview of Ph. infestans ABC transporter expression

We generated a heatmap that shows the expression of ABC transporters in Ph. infestans during its interaction with plants in the early, middle and late stage, which respectively represent the stages of initial infection, engraftment success, and killing host cells [22–23] (Fig. 9). The middle stage and late stage samples clustered with each other, separated from the early stage samples. On the whole, most ABC proteins of Ph. infestans were expressed in the middle and late stages. The sole exception was ABCB proteins, which had high expression in the early stage, except for XP_002899791.1, XP_002998888.1, and XP_002897125.1. Notably, XP_002897125.1, which belong to the half-size ABCB group II members of which are close to bacterial ABC transporters, was highly expressed in the middle stage. Most ABCA proteins were very highly expressed in the middle stage, except XP_002895909, which was expressed in the early stage, and XP_002909428.1, which was expressed in the late stage. Of note, XP_002909428.1 belongs to the ABCA group III, which are close to bacterial multi-drug transporters. There were seven filleted ABCC proteins expressed very highly in the middle stage, and XP_00298372.1 and XP_002908040.1 were expressed in the late stage. XP_002996976.1, an ABCC protein, was not expressed during the interaction of Ph. infestans and plant. The expression of ABCD transporters (except XP_002895826.1) went up and then down and peaked in the middle stage. There were a few ABCG proteins expressed in the early stage, but not specifically. Most ABCG proteins were expressed both in the middle and late stages, with only the intensity of expression being different, and ABCG have broad substrate specificity. Of note, there were six ABCG members (XP_002904653.1, XP_002904654.1, XP_002904655.1, XP_002904843.1, XP_002998407.1, and XP_002899779.1) specifically expressed in the late stage.

The ABCA subfamily

ABCA proteins are common in eukaryotes and prokaryotes but they lack a structural counterpart in yeast [11.24-28]ABCA members have previously been implicated in cellular lipid efflux and high-density lipoprotein metabolism[29.30]. For example, ABCA of Leishmania tropica is expressed mainly in the flagella and can regulate membrane movements such as endocytosis, exocytosis, and vesicle trafficking [31]. In particular, overexpressing ABCA1 can reduce the infectivity of Leishmania [31]. In Staphylococcus aureus, ABCA not only has a role in drug efflux but can also secrete phenol-soluble modulins and contribute to S. aureus virulence [27].

In our study, ABCA proteins showing the multi-line type, and this finding was similar with the result of Morris et al. for the ABCA transporter subfamily of Ph. ramorum and Ph. sojae, which were regarded as having different evolutionary patterns during their long-term evolution [26]. Meanwhile, there were absented the ABCA proteins in a few species, proposing multiple loss events during evolution and that some ABCA proteins are not essential for viability. Of course, it could also that the lost proteins were incomplete ABC proteins that were screened out. The phylogenetic analysis of ABCA subfamily results shows that the ABCA1 group I and II were closer to the H. sapiens and plants. In H. sapiens, ABCA1 mutation may cause Tangier disease (TD), which results from cholesterol accumulation in various tissues [7]. Therefore, the ABCA group I and II proteins may play a role in lipid efflux in oomycetes. The phylogenetic relationship of group III (XP_002909428.1) was closer to bacteria. The ABCA proteins of bacteria in this study play a role in multi-drug resistance and efflux of virulence factors [27]. And ABCA group III proteins may participate in interactions with plants by translating virulence factors from oomycetes to hosts. But the exact transport function is unknown and needs experimental verification. Furthermore, we found ABC-2 type ABC transporters in the ABCA subfamily which transport polysaccharides. Lipopolysaccharide is a key substance that causes immune responses in plants. ABCA proteins, which are highly expressed in the middle stage, also could further explain why this subfamily is related to immunity during host oomycete interactions.

The ABCB subfamily

The ABCB subfamily is a large class of ABC proteins and consists of full-length and half-size members that are widely distributed in eukaryotes and are functionally diverse. It includes multi-drug resistance (MDR) proteins [32], heavy metal resistance[8], P-glycoproteins (PGP)[33], antigen processing [34], mitochondrial peptide and pheromone export[35], and Fe/S cluster proteins biogenesis[36]. In our study, the ABCB proteins in oomycetes mainly contained MdIB, Atm1, and PGP functional domains. MdIB and Atm1 are protective proteins against host plant toxins and heavy metals in the environment; for example, VerA in Clonostachys rogersoniana, which is an MdIB transporter, and Atm1 in Novosphingobium aromaticivorans [37.38] In plants, ABCB proteins have been shown to be involved in the transport of both growth regulators (auxin) and secondary metabolites, and the plant secondary metabolites participate in deterring herbivory and resistance to pathogens[39]. At the same time, ABCB proteins play a role in absorption. OsABCB14 in Oryza sativa (rice) has a role as a plasma membrane auxin influx transporter and is also involved in ion homeostasis [40].

In our study, the ABCB proteins in oomycetes mainly contained MdIB, Atm1, and PGP functional domains. MdIB and Atm1 are protective proteins against host plant toxins and heavy metals in the environment; for example, VerA in C. rogersoniana, which is an MdIB transporter, and Atm1 in N. aromaticivorans [37.38] The phylogenetic results of ABCB full-length and ABCB half-size transporters is consistent with the homologous relationship for the ABCB subfamily of Ph. ramorum and Ph. sojae by Morris et al. (2009), suggesting that the ABCB subfamily have a more diverse evolutionary pattern. Meanwhile, ABCB full-length of group II and III branches together, may have the same or similar functions. However, the ABCB full-length group IV proteins is different from group II and III, but closer to bacteria (MAD20250.1). We were speculated Group IV genes might be horizontally transferred from prokaryotes during species differentiation and the proteins that have been horizontally transferred are often related to pathogenicity. In Bacteria ABCB group, the PKB66159.1 was annotated as CydC in bacteria. CydC is required for survival and virulence in Brucella abortus, and its absence results in severe virulence attenuation [41]. This also indicates that group IV may be related to oomycete pathogenicity. In addition, the ABC-B half-size proteins in oomycetes are homologous with ABCB10, ABCB25 and ABCB7. ABCB10 can increase mitochondrial reactive oxygen species production and oxidative stress [42]. The production of reactive oxygen species in plants can cause hypersensitivity in cells and the subsequent death of the pathogen (HR response). ABC Atm, ABCB25, and ABCB7 are required for iron-sulfur (Fe S) cluster biosynthesis outside of mitochondria [43.44]. Although we do not know the exact function of bacterial ABCB besides oomycete ABCB II, it is possible that group II may have been transferred to other groups from bacteria. Moreover, in combination of us analyze the ABCB proteins transporter expression, which are highly expressed in the early stage, suggested that some ABCB proteins might be a plasma membrane influx transporter.

The ABCC subfamily

Different from other ABC subfamilies, most ABCC proteins are full-length, and many proteins have an N-terminal hydrophobic region [24]. ABCC transporters also constitute a class of MRPs. The mechanism of tolerance and resistance of toxic substances is by sequestering toxic hydrophobic compounds into specialized designated organelles, or by directing them for secretion. For example, ABCC proteins can conjugate the formation of glutathione–Pb (GS-Pb) to vacuoles for Pb detoxification in yeast [45]. In pathogens, although most ABCC proteins are multi-drug resistance-associated members, only a few proteins are related to pathogenicity. ABC2 in M. grisea is required for multidrug resistance but is not involved in pathogenicity [46]. And ABC5 of the ABCC subfamily in M. grisea is responsible for the efflux of phytotoxic metabolites produced upon interacting with rice leaves, and if lost will reduce pathogenicity [47].

In the present result, there are three orthologous groups in group I and two orthologous groups in group V. Although there are differences in the protein sequences of different orthologue, they can still cluster in the phylogenetic tree. This shows that they may retain similar functions. At the same time, there were no ABCC proteins in obligate biotroph oomycetes from group I and V, indicating that biotrophic oomycetes have experienced multiple loss events during evolution of the ABCC group I and V. It further demonstrates that the two groups of ABCC proteins are not essential for biotrophic oomycete viability. There are only partial species in groups III, IV, and VI, which may be related to their unique biology. ABCC group II clustered with YCF1 and MRP1 with a high bootstrap value. YCF1 in fungi excretes cadmium and is localized on the vacuolar membrane [35]. MRP1 and YCF1 are almost identical, but the types of heavy metals excreted by MRP1 and YCF1 are different [36]. This shows that ABCC group II in oomycetes may also act as transporters that excrete heavy metals. ABCC group IV clustered with the plant ABCC13 and human MRP7 with a high bootstrap value, indicating that they are nearly identical. ABCC13 is a protein that participates in heavy metal efflux in plants [38]. MRP7 is a lipophilic anion pump translating glucuronic acid as a drug-resistant transporter [37]. Although group II and IV have different evolutionary relationships, they are both involved with heavy metal efflux. In addition, most ABCC proteins expressed very highly in the middle stage and the late stage. Thus, we believe that ABCC proteins appear to be uneven in their evolutionary distribution and species diversity. These results also indicate that ABCC proteins are involved in the translation of distinctive secondary metabolites or exogenous toxins or as a transporter of participate in heavy metal efflux rather than in basal metabolism.

The ABCD subfamily

In our result, all the ABCD proteins are half-size and not present in every oomycete species. The ABCD proteins can be partially or completely lost over the long evolution histories of oomycetes. Also, the function of ABCD in oomycetes may be unique. Interestingly, we found RxLR and RxLR-like residues in ABCD proteins. RxLR is a virulence factor in oomycetes, and this motif is present in secreted proteins and in some has been demonstrated to facilitate the uptake of proteins into the host cytoplasm, which can increase plant susceptibility. Meanwhile, ABCD proteins of oomycetes did not cluster with any proteins from fungi or bacteria, showing that oomycete ABCD proteins do not have a special role in pathogens. In short, ABCD proteins are also located in peroxisomes and can participate in fatty acids (FA) export in all oomycetes. It’s worth noting that FA is plays a regulatory role in plant pathogen interactions and stress network signaling, so ABCD, which exports FA, deserves attention in plant oomycete interactions. Moreover, the expression of ABCD transporters went up and then down and peaked in the middle stage. However, owing to the ABCD proteins data partially or completely lost, we cannot speculate much more on its function, more research is needed.

The ABCG subfamily

The Pleiotropic Drug Resistance (PDR)/ABCG subfamily is ubiquitous throughout humans, plants, animals, fungi, and oomycetes, but have yet to be found in bacteria [26.48-50] The ABCG subfamily is composed of two major types of proteins: half-size and full-length. The special feature of ABCG proteins that makes them different from other ABC is was their revise topology, with the nucleotide-binding domain preceding the transmembrane domain. Half-size ABCG proteins only contain one NBD and TMD, and full-length ABCG proteins contain two NBD and TMD. Full-length PDR transporters are mainly present in plants, fungi, and probably evolved via duplication of half-sized ABCG before the separation of fungi and plants [51]. PDR proteins are widely distributed membrane proteins that play a role in extruding a variety of xenobiotics. For example, ABCG2 in humans exhibits broad substrate specificity to xenobiotic compounds and confers cancer cells resistance to anticancer drugs [52]). In oomycetes, there are also many half-size ABCG proteins that have NBD copy event. Oomycete ABCG are highly diverse, and their functions also vary during different stages of infection.

In this study, all Phytophthora and Peronospora clustered together with ABC2, E2 and PDR1 (group II-VI), indicating that Pythium, Albugo, Aphanomyce, and Saprolegnia may have broader special substrate functions. ABC2 is the first ABC transporter found in M. grisea, which is related to fungal multidrug sensitivity, but not to controlled spore bourgeon and restrained accrete-spore [53]. Dihydrolipidamide acetyltransferase (E2) is a protein with immunogenic activity. It has been found in bacteria and is considered to be a vaccine candidate [54]. The PDR1 of plants is localized on the plasma membrane and is resistant to Phytophthora [50]. ABCG proteins may increase sensitivity of plants to disease, but it is unclear if they are related to host cell death. Oomycetes ABCG VII clustered with H. sapiens ABCG1, which is an eye pigment transporter. Therefore, we can surmise that ABCG group VII transporter genes were created through a half-size horizontal transfer to oomycetes and then had a duplication event. Only Phytophthora and Pythium were involved in the separated group (group IV), indicating that group IV may be involved in causing host cell death. Furthermore, Most ABCG proteins were expressed both in the middle and late stages, with only the intensity of expression being different, which showed that ABCG have broad substrate specificity. Of note, there were six ABCG members specifically expressed in the late stage, indicating that these ABCG proteins may transport effectors that cause plant cell death in the necrotrophic phase. Therefore, we inferring the main ABC proteins that participate in the necrotrophic phase are ABCG proteins.

Our analysis of oomycete ABC proteins provides insight into the diversity of the five subfamilies among the main oomycete lineages. ABC proteins are firmly established as key players of cellular processes involved with xenobiotic detoxification and disease resistance, especially in plant pathogen interactions. The architectural feature of oomycete ABC proteins suggests that their binding sites may not be limited to ATP, but also bind metals and other chemicals. And transmembrane domain also may not be limited to one or two, but also three, four, or more. In this study, 1303 ABC proteins were identified, and 965 were filtered in a phylogenetic analysis from 21 species of oomycete representing three different lifestyles (biotrophy, hemibiotrophy and necrotrophy). Moreover, we ascertained that ABCG proteins are the largest group of ABC proteins in most oomycete species analyzed, although ABCC had the most members in some oomycete species. Phylogenetic analysis allowed us to trace the evolutionary history of oomycete ABCs, which shows evidence of eukaryotic microorganism diversification. By analyzing the expression data of Ph. infestans ABC proteins filtered in this study, we were able to provide the putative role of these proteins and candidate avirulence or virulence factors. The result of this work offers useful information that could help, for example, to discover more or broader special roles of ABC proteins in plant or animal pathogenesis, and help to address several biological questions concerning the evolution of the relationships between oomycete genomes of different species.

Identification of ABC transporters in oomycetes

Twenty-one oomycete genomes along with annotations and reference data were obtained from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) database and Ensembl Protists database (https://protists.ensembl.org/index.html) (Table 2). Ph. infestans ABC transporter protein sequences were identified using Unix from annotated gff files. Twenty-two oomycete ABC protein sequences were identified by BLAST with ABC proteins of Ph. infestans searches on oomycete genomes, which were executed using an E-value of 10 − 4 as a cut-off [55]. Transmembrane predictions were performed with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) [56], and ATP binding domain predicted by NCBI-conserved domain search service tool (CDD). Orthofinder v2.2.1 [57] was used to find proteins orthologous to ABC proteins across the 21oomycete species. Consensus domain architecture for each orthogroup was defined, and proteins that deviated from this consensus were subjected to manual inspection. We used Ph. infestans as a BLAST reference sequence to confirm the separated orthologous ABC proteins subfamilies from NCBI to allow the fast assignment of the analyzed sequences to a particular subfamily.

Table 2

Published oomycete genomes.
Species Name	Genome available link
Albugo candida	http://protists.ensembl.org/Albugo_candida/Info/Index
Albugo laibachii	http://protists.ensembl.org/Albugo_laibachii/Info/Index
Aphanomyces astaci	http://protists.ensembl.org/Aphanomyces_astaci/Info/Index
Aphanomyces invadans	http://protists.ensembl.org/Aphanomyces_invadans/Info/Index
Bremia lactucae	https://www.ncbi.nlm.nih.gov/assembly/GCA_004359215.1/
Hyloperonospora arabidopsidis	http://protists.ensembl.org/Hyaloperonospora_arabidopsidis/Info/Index
Peronospora effusa Race 13	https://www.ncbi.nlm.nih.gov/assembly/GCA_003843895.1
Peronospora effusa Race 14	https://www.ncbi.nlm.nih.gov/assembly/GCA_003704535.1
Peronospora tabacina J2	https://www.ncbi.nlm.nih.gov/assembly/GCA_003843895.1
Peronospora tabacina S26	https://www.ncbi.nlm.nih.gov/assembly/GCA_002099245.1
Plasmopara halstedii	http://protists.ensembl.org/Plasmopara_halstedii/Info/Index
Phytophthora infestans	http://protists.ensembl.org/Phytophthora_infestans/Info/Index
Phytophthora sojae	http://protists.ensembl.org/Phytophthora_sojae/Info/Index
Phytophthora ramorum	http://protists.ensembl.org/Phytophthora_ramorum/Info/Index
Pythium aphanidermatum	http://protists.ensembl.org/Pythium_aphanidermatum/Info/Index
Pythium arrhenomanes	http://protists.ensembl.org/Pythium_arrhenomanes/Info/Index
Pythium irregular	http://protists.ensembl.org/Pythium_irregulare/Info/Index
Pythium iwayamai	http://protists.ensembl.org/Pythium_iwayamai/Info/Index
Pythium ultimum	http://protists.ensembl.org/Pythium_ultimum/Info/Index
Saprolegnia diclina vs20	http://protists.ensembl.org/Saprolegnia_diclina_vs20/Info/Index
Saprolegnia parasitica CBS 223.65	http://protists.ensembl.org/Saprolegnia_parasitica_cbs_223_65/Info/Index

Phylogenetic analysis of ABC transporters

To study the variability of the deduced amino acid sequences of ABC proteins in oomycetes, the identified ABC proteins were aligned using MAFFT software [55]. A phylogenetic tree was constructed using maximum likelihood method with the RAxML v8.2.9[58] and MEGA program. And the confidence of the tree topology was assessed through a bootstrap analysis of 1000 replicates. Alignments and trees were visualized using Geneious version R11[59]. Detailed analyses were performed separately for each subfamily, and in the case of ABCA proteins, we used all full-length proteins (the full-length proteins contained at least nine transmembrane helices); in the case of ABCB proteins, we used full-length and half-size proteins; in the case of ABCC proteins, we used all full-length proteins; in the case of ABCD proteins, the smallest ABC subfamily, we used all proteins; in the case of ABCG proteins, because the ABCG subfamily is the biggest, we only studied the full-length proteins. To analyze the evolution of ABC transporters in oomycetes, we added H. sapiens, plant, fungal, and bacterial ABC transporters from NCBI using Ph. infestans as a BLAST reference sequence.

Expression profiling of ABC transporters using Ph. infestans

Previously published Ph. infestans expression data were used to determine the expression patterns of ABC transporter genes at different infection stages. RNA-seq analysis was performed using early (36 h), middle (60 h), and late stage (96 h) interactions between Ph. infestans and tomato tubers [60]. The reads were mapped to their respective assembly genome using STAR v2.60c[61]. Trimmed Mean of M (TMM) preserve the mapped reads data by introducing factors as offsets in the statistical model [62]. The RNA-seq data were analyzed using R statistical software [63]. A heatmap for infection stage-specificity of the expression of ABC transporter proteins of Ph. infestans used several R packages, including tidyverse, ggplot2[64], gplots[65], edgeR[66], and RColorBrewer [67].

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) and Ensembl Protists database (https://protists.ensembl.org/index.html)

Competing interests

The authors declare that they have no competing interests.

Funding

The research was funded by the Fundamental Research Funds for the Central Universities, Southwest Minzu University (No. ZYN2022019). This work was supported by Open Fund of Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu University), Ministry of Education (KF2021010).

Author contribution

LYX and ZY carried out the molecular genetic studies and drafted the manuscript. CYJ participated in the design of the study, YRW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgments

YXL and YZ gratefully acknowledge the China Scholarship Council (CSC) for the Ph.D. scholarship.

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No competing interests reported.

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Genome-wide identification, phylogenetic analysis, and expression profiling of ATP-binding cassette transporters in oomycetes

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Abstract

Figures

Background

Results

Identification of oomycete ABC transporters

Phylogenetic analysis of ABCA subfamily

Phylogenetic analysis of multi-drug resistance (MDR)/ABCB subfamily

Phylogenetic analysis of MRP/ABCC subfamily

Phylogenetic analysis of ABCD subfamily

Phylogenetic analysis of ABCG subfamily

Overview of Ph. infestans ABC transporter expression

Discussion

The ABCA subfamily

The ABCB subfamily

The ABCC subfamily

The ABCD subfamily

The ABCG subfamily

Conclusions

Materials And Methods

Identification of ABC transporters in oomycetes

Phylogenetic analysis of ABC transporters

Expression profiling of ABC transporters using Ph. infestans

Declarations

References

Additional Declarations

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