**1. Introduction**

The exocyst complex is an evolutionarily conserved octameric tethering factor, which mediates the fusion of post-Golgi secretory vesicle with the plasma membrane (PM) and plays a major role in exocytosis [1,2]. EXO70 is a key member of the exocyst complex and has been found to be widely present in yeast, mammals and plants [3]. In yeast and mammals, the *EXO70* only has a single copy, while plants have multiple copies of *EXO70* genes [4] ranging from 21 to 47 *EXO70* members in potatoes (*Symphytum tuberosum*), *Arabidopsis*, *Populus trichocarpa* and rice [1,5,6]. The *EXO70s* of land plants possibly originated from three ancient *EXO70* genes and thus can be divided into three groups

*EXO70.1, EXO70.2* and *EXO70.3*. They have been further duplicated independently in the moss, lycophyte and angiosperm lineages, and in the subsequent lineage-specific multiplications which are represented by nine subgroups (*EXO70A-EXO70I*) [4,7–9].

The function of *EXO70* has been extensively studied in yeast, and mammals [3,10–15]. In plants, *EXO70s* have been proven to play diverse roles in regulating plant growth and coping with adverse biotic/abiotic stresses. In *Arabidopsis*, *EXO70A1* has been implicated in a wide range of developmental processes, including the differentiation of tracheary elements [16,17], the development of seed coat, root hair and stigmatic papillae [18], the recycling of the auxin efflux carrier proteins (PIN1 and PIN2) [19] and the formation of the Casparian strip [20]. *EXO70C1* and *EXO70C2* regulated the polarized growth and maturation of the pollen tube [21,22]. *EXO70H4* regulates trichome cell wall maturation by mediating the secretion and accumulation of callose and silica [23,24]. The rice *OsEXO70A1* is necessary for vascular bundle differentiation and assimilation of mineral nutrients [5]. The legumes EXO70J7, EXO70J8 and EXO70J9 are members of an atypical subgroup of EXO70 proteins (EXO70J) that regulate leaf senescence and nodule formation [25]. In *Nicotiana benthamiana*, silencing all the paralogue genes in subgroups EXO70A (six), C (three), D (four) and G (six) resulted in a smaller leaf phenotype [6].

Evidence has accumulated for the critical role of *EXO70s* in plant-pathogen interactions or responses to abiotic stresses. In *N. benthamiana*, the silencing of two *EXO70B* paralogues led to increased susceptibility to *Phytophthora infestans* [6]. Three of the 23 members of the *Arabidopsis EXO70* gene family (*EXO70B1*, *EXO70B2* and *EXO70H1*) have been proven to be involved in plant immunity [26]. *AtEXO70B1* and *AtEXO70B2* belong to the same subgroup and are both involved in plant immunity, of which *AtEXO70B1* a negative regulator, while *AtEXO70B2* is a positive regulator. *AtEXO70B1* underwent autophagic transport, and the loss-of-function of *exo70B1* led to reduced numbers of internalized autophagosomes, accumulation of salicylic acid (SA), and finally, ectopic hypersensitive responses and enhanced resistance to several pathogens. AtEXO70B1's regulation of disease resistance, either by interacting with TIR-NBS2, a truncated version of the classical nucleotide binding (NB) domain and a leucine-rich repeat (LRR)-containing (NLR) intracellular immune receptor-like protein [27–29], or by interacting with RIN4, a well-known regulator of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) [29]. AtEXO70B2 regulated innate immunity via interacting with a negative PTI regulator, AtPUB22, which mediated the ubiquitination and degradation of AtEXO70B2 and contributed to PTI. The *exo70B2* mutants showed aberrant papillae with halos and were susceptible to different PAMPs and pathogens [30,31]. *AtEXO70B1* and *AtEXO70B2* also contribute to the abiotic stress response; both were positive regulators of stomatal movement. The response to mannitol (drought) treatments is in either an abscisic acid (ABA)-dependent or -independent manner [32,33]. *AtEXO70H1* is a homolog of *AtEXO70B2* and is also involved in plant immunity [31]. Three of the 47 *EXO70* members of rice (*OsEXO70E1*, *OsEXO70F2* and *OsEXO70F3*) were reported to participate in plant immunity [5]. OsEXO70E1 is attributed to planthopper resistance by interacting with a broad resistance protein, *Bph6*. Interaction of the two proteins increased exocytosis and blocked the feeding of a planthopper by cell wall thickening at the infection sites [34]. The importance of *EXO70* in plant immunity was also shown by the fact that some of the *EXO70s* were targets of the secreted effectors of the plant pathogen. Both OsEXO70F2 and OsEXO70F3 were targets of the *Magnaporthe oryzae* effector AVR-Pii, and OsEXO70F3 was proven to play an important role in *Pii*-dependent resistance by interacting with AVR-Pii [35].

Accumulated evidence has shown that a large number of *EXO70s* exist in plants; however, only a few have had their biological roles elucidated [5,35,36]. Due to the huge genome size and complexity [37], the knowledge of the *EXO70* gene family from the *Triticeae* species is rather limited. In the last five years, the genome sequences of wheat and its ancestor species have been released, which makes genome-wide identification of a gene family in the *Triticeae* species feasible [38–43].

species feasible [38–43].

*Haynaldia villosa* L. (2*n* = 2x = 14, VV) is a diploid wild relative of wheat. Previous studies showed that *H. villosa* is a valuable genetic resource harboring many elite traits, such as resistance to several wheat diseases and tolerance to abiotic stresses [44–46]. In the present study, different members of the *EXO70* gene family are identified by browsing the released genome sequences of the *Triticea* species. Specific primer pairs are designed, *EXO70s* are cloned from *H. villosa* and their potential functions are elucidated by expression profiles based on in silico analysis and quantitative RT-PCR (qRT-PCR). The obtained results would help us to understand the evolution and diversification of the *EXO70s* among *Triticeae* species and their potential roles in plant immunity and responses to abiotic stresses. study, different members of the *EXO70* gene family are identified by browsing the released genome sequences of the *Triticea* species. Specific primer pairs are designed, *EXO70s* are cloned from *H. villosa* and their potential functions are elucidated by expression profiles based on *in silico* analysis and quantitative RT-PCR (qRT-PCR). The obtained results would help us to understand the evolution and diversification of the *EXO70s* among *Triticeae* species and their potential roles in plant immunity and responses to abiotic stresses. **2. Results** 

*Int. J. Mol. Sci.* **2018**, *19*, x 2 of 22

Accumulated evidence has shown that a large number of *EXO70s* exist in plants; however, only a few have had their biological roles elucidated [5,35,36]. Due to the huge genome size and complexity [37], the knowledge of the *EXO70* gene family from the *Triticeae* species is rather limited. In the last five years, the genome sequences of wheat and its ancestor species have been released, which makes genome-wide identification of a gene family in the *Triticeae*

*Haynaldia villosa* L. (2*n* = 2x = 14, VV) is a diploid wild relative of wheat. Previous studies

resistance to several wheat diseases and tolerance to abiotic stresses [44–46]. In the present

#### **2. Results** *2.1. Identification and Phylogenetic Relationship Analysis of the EXO70 Gene Family in Triticeae Species*

#### *2.1. Identification and Phylogenetic Relationship Analysis of the EXO70 Gene Family in Triticeae Species* In total, 200 *EXO70* genes were identified from the public database of five *Triticeae* species.

In total, 200 *EXO70* genes were identified from the public database of five *Triticeae* species. Among them, there were 26 each from *T. urartu*, *Ae. Tauschii* and *H. vulgare*; 47 from *T. dicoccoides*; and 75 from common wheat (*T. aestivum)*, respectively. Fifteen *EXO70s* from *H. villosa* were obtained by homology cloning (Figure 1a). The evolutionary relationship of the above 215 *Triticeae EXO70s*, along with 22 from *Brachypodium distachyon*, 41 from rice and 23 from *Arabidopsis*, were phylogenetically analyzed (Figure 1b, Table S1). These *EXO70s* were divided into three major groups, *EXO70.1*, *EXO70.2* and *EXO70.3*, which were further assigned to nine subgroups, from *EXO70A* to *EXO70I*, according to a phylogenetic tree (Figure 1b). The *EXO70A* subgroup belongs to the group *EXO70.1*, in which 43 (14.28%) *EXO70s* were included; the EXO70B, C, D, E, F, H, and I subgroups belong to the group *EXO70.2*, in which 229 (76.08%) *EXO70s* were included; the *EXO70G* subgroup belongs to the group *EXO70.3*, in which 29 (9.63%) *EXO70s* were included. The *EXO70I* subgroup has the most members (74, 24.58%), followed by *EXO70F* (48, 15.95%) and *EXO70A* (43, 14.28%) (Figure 1c). Based on the subgroups and genome allocation, the wheat *EXO70s* were designated [7,47]. For example, the *EXO70B1* from *T. dicoccoides* located on chromosome 1A was assigned *TdEXO70B1-1A*. In *Arabidopsis*, the *EXO70I* subgroup is missing, whereas the *EXO70I* subgroup in *Triticeae* species appeared to be the most divergent. However, our analysis led to a new insight: the *EXO70I* subgroup belongs to *EXO70.2*, rather than *EXO70.3* from other species [36] (Figure 1b,c). Among them, there were 26 each from *T. urartu*, *Ae. Tauschii* and *H. vulgare*; 47 from *T. dicoccoides*; and 75 from common wheat (*T. aestivum)*, respectively. Fifteen *EXO70s* from *H. villosa* were obtained by homology cloning (Figure 1a). The evolutionary relationship of the above 215 *Triticeae EXO70s*, along with 22 from *Brachypodium distachyon*, 41 from rice and 23 from *Arabidopsis*, were phylogenetically analyzed (Figure 1b, Table S1). These *EXO70s* were divided into three major groups, *EXO70*.*1*, *EXO70*.*2* and *EXO70*.*3*, which were further assigned to nine subgroups, from *EXO70A* to *EXO70I*, according to a phylogenetic tree (Figure 1b). The *EXO70A* subgroup belongs to the group *EXO70.1*, in which 43 (14.28%) *EXO70s* were included; the EXO70B, C, D, E, F, H, and I subgroups belong to the group *EXO70*.2, in which 229 (76.08%) *EXO70s* were included; the *EXO70G* subgroup belongs to the group *EXO70*.3, in which 29 (9.63%) *EXO70s* were included. The *EXO70I* subgroup has the most members (74, 24.58%), followed by *EXO70F* (48, 15.95%) and *EXO70A* (43, 14.28%) (Figure 1c). Based on the subgroups and genome allocation, the wheat *EXO70s* were designated [7,47]. For example, the *EXO70B1*  from *T. dicoccoides* located on chromosome 1A was assigned *TdEXO70B1-1A*. In *Arabidopsis*, the *EXO70I* subgroup is missing, whereas the *EXO70I* subgroup in *Triticeae* species appeared to be the most divergent. However, our analysis led to a new insight: the *EXO70I* subgroup belongs to *EXO70*.*2*, rather than *EXO70*.*3* from other species [36] (Figure 1b,c).

**Figure 1.** *Cont.*

**Figure 1.** The number and phylogenetic relationships of *EXO70* family genes from *T. aestivum*, *T. urartu*, *Ae. tauschii*, *T. dicoccoides*, *H. vulgare*, *A. thaliana*, *Oryza sativa*, *Brachypodium distachyon* and *H. villosa*. (**a**) The total number of *EXO70* gene family in nine species. (**b**) The phylogenetic tree of nine *Triticeae* species. Species abbreviations: Ta, *Triticum aestivum*; Tu, *Triticum urartu*; Aet, *Aegilops tauschii*; Td, *Triticum dicoccoides*; Hv, *Hordeum vulgare*; At, *Arabidopsis thaliana*; Bd, *Brachypodium distachyon*; Os, *Oryza sativa*; -V, *Haynaldia villosa*. (**c**) The number of the *EXO70* gene family from nine species in each of the subgroups. The horizontal/longitudinal coordinate axis represents the number of genes and different subgroups, respectively. **Figure 1.** The number and phylogenetic relationships of *EXO70* family genes from *T. aestivum*, *T. urartu*, *Ae. tauschii*, *T. dicoccoides*, *H. vulgare*, *A. thaliana*, *Oryza sativa*, *Brachypodium distachyon* and *H. villosa*. (**a**) The total number of *EXO70* gene family in nine species. (**b**) The phylogenetic tree of nine *Triticeae* species. Species abbreviations: Ta, *Triticum aestivum*; Tu, *Triticum urartu*; Aet, *Aegilops tauschii*; Td, *Triticum dicoccoides*; Hv, *Hordeum vulgare*; At, *Arabidopsis thaliana*; Bd, *Brachypodium distachyon*; Os, *Oryza sativa*; -V, *Haynaldia villosa*. (**c**) The number of the *EXO70* gene family from nine species in each of the subgroups. The horizontal/longitudinal coordinate axis represents the number of genes and different subgroups, respectively.

The 15 cloned *EXO70s* from *H. villosa* were designated as *EXO70A1*-*V* to *EXO70I1*-*V* according to the phylogenetic relationship to wheat *EXO70s*. They belong to nine subgroups, three each to *EXO70A* and *F*, two each to *EXO70D* and *G* and one each to *EXO70B*, *C*, *E*, *H* and *I* (Figure 1). Their CDS length ranges from 801 bp (*EXO70H1-V*) to 2007 bp (*EXO70C1-V*) and their isoelectric point varies from 4.52 (*EXO70B1-V*) to 10.19 (*EXO70G1-V*) (Table 1). The 15 cloned *EXO70s* from *H. villosa* were designated as *EXO70A1*-*V* to *EXO70I1*-*V* according to the phylogenetic relationship to wheat *EXO70s*. They belong to nine subgroups, three each to *EXO70A* and *F*, two each to *EXO70D* and *G* and one each to *EXO70B*, *C*, *E*, *H* and *I* (Figure 1). Their CDS length ranges from 801 bp (*EXO70H1-V*) to 2007 bp (*EXO70C1-V*) and their isoelectric point varies from 4.52 (*EXO70B1-V*) to 10.19 (*EXO70G1-V*) (Table 1).

**Table 1**. *EXO70* genes cloned from *H. villosa.* 

**Number Name ORF (bp) AA (aa) DL (aa) PI MW (KD)**  1 EXO70A1-V 1914 637 268–623 7.71 71.48 2 EXO70A2-V 1944 647 273–631 8.78 73.07 3 EXO70A3-V 882 294 1–283 8.83 32.61


**Table 1.** *EXO70* genes cloned from *H. villosa*. *Int. J. Mol. Sci.* **2018**, *19*, x 4 of 22

Abbreviations: ORF, open reading frame; AA, amino acids; DL, PFam03081 domain location; PI, protein isoelectric point; MW, protein molecular weight. PI, protein isoelectric point; MW, protein molecular weight.

DNAMan was used to explore the amino acid sequence feature of *H. villosa EXO70s*. The

DNAMan was used to explore the amino acid sequence feature of *H. villosa EXO70s*. The pfam03081 domain at the C-terminus was relatively conservative, shown by the fact that all of the *EXO70s* had the same amino acid in the 11 sites (in the red rectangle in Figure 2a). R programming language was used to visualize the sequence similarities. The same subgroups shared more common sequences, e.g., *EXO70D1-V* and *EXO70D2*-*V* shared about 80% similarity, while different subgroups had low sequence similarities, e.g., *EXO70H1-V* and *EXO70I1-V* only had 21.46% similarity (Figure 2b). pfam03081 domain at the C-terminus was relatively conservative, shown by the fact that all of the *EXO70s* had the same amino acid in the 11 sites (in the red rectangle in Figure 2a). R programming language was used to visualize the sequence similarities. The same subgroups shared more common sequences, e.g., *EXO70D1-V* and *EXO70D2*-*V* shared about 80% similarity, while different subgroups had low sequence similarities, e.g., *EXO70H1-V* and *EXO70I1-V* only had 21.46% similarity (Figure 2b).

**Figure 2.** The amino acid sequence feature analysis of *EXO70* gene in *H. villosa*. (**a**) The amino acid sequence of pfam03081 domain for each *EXO70* was selected for multiple sequence alignment analysis by DNAMan. The 11 identical amino acids were indicated in red frame. The three colors of black, red and blue represent the level of similarity of amino acids, from high to low. (**b**) Sequence similarity analysis using the R programming language. The color scale bar represents sequence similarity between different genes. Red and yellow indicate that the sequence similarity was greater than 80% and 60%, respectively. Blue indicates that the **Figure 2.** The amino acid sequence feature analysis of *EXO70* gene in *H. villosa*. (**a**) The amino acid sequence of pfam03081 domain for each *EXO70* was selected for multiple sequence alignment analysis by DNAMan. The 11 identical amino acids were indicated in red frame. The three colors of black, red and blue represent the level of similarity of amino acids, from high to low. (**b**) Sequence similarity analysis using the R programming language. The color scale bar represents sequence similarity between different genes. Red and yellow indicate that the sequence similarity was greater than 80% and 60%, respectively. Blue indicates that the sequence similarity was less than 40%.

sequence similarity was less than 40%.
