*3.3. Function Conserve or Differentiation of the EXO70 Gene Family in Common Wheat and H. villosa*

An orthologous gene is one that diverged after evolution to give rise to different species; this gene generally maintains a similar function to that of the ancestral gene that it evolved from [67]. In our study, some *EXO70* orthologous genes from *H. villosa* exhibited a similar expression pattern to

common wheat. For example, *EXO70A3-V* and *TaEXO70A3* showed a high expression level under heat stress; *EXO70B1-V* was preferred to *TaEXO70B1-3AL*, which was induced by *Bgt* at a late stage (48 h), but not by drought and heat stress; *EXO70E1-V* and *TaEXO70E1-3B* were upregulated by *Bgt* treatment; the expression of *EXO70H1-V* and *TaEXO70H1-1DL* was increased in response to heat (Figures 5 and 6). Therefore, it is reasonable to presume that some *EXO70* genes from *H. villosa* may have a similar function to the corresponding *EXO70* genes from common wheat. In plants, *AtEXO70B1*, *AtEXO70B2*, *AtEXO70H1* and *OsEXO70E1* are known for their roles in innate immunity [6,27–31, 34]. In the literature, genes such as *TaEXO70B1*/*B2* (with their homologous alleles), *EXO70B1-V*, *TaEXO70E1-3B*, *EXO70E1-V*, *TaEXO70H1-1DL* and *EXO70H1-V* were induced by *Pst*/*Bgt* treatment. Thus, we hypothesize that those genes also play an important role in plant defense responses and it is worth conducting further study to prove their function.

The long-term evolutionary fate of paralogous genes will still be determined by functions, with the genes that appeared to be sub-functionalized or neo-functionalized probably having higher rates of gene birth because of the increased adaptability. In contrast, the functional redundancy gene is unlikely to be stably maintained in the genome [55,57,68,69]. Paralogous/orthologous genes may diverge in expression to achieve more complex control of the same genetic network, balancing the relationship between internal growth and external environmental stimuli so that they "pay" the least and get the most [70]. In *Arabidopsis*, *EXO70C1*/C2 were involved in pollen development and mainly localized pollen related tissue [21,22]. In common wheat, six *TaEXO70C* genes had an undetectable expression level in roots/stem/leaves (Figure 5a), but *EXO70C1-V* showed a moderate expression level in the stem and responded to drought and ABA treatment (Figure 6). Studies have shown that ABA has contributed to osmotic stress tolerance by regulating stomatal aperture and guard cells [71]. Therefore, *EXO70C1-V* perhaps plays an important role in drought tolerance. In rice, OsExo70F3 interacts with AVR-Pii and plays a crucial role in triggered immunity [35]. In our research, including the copy number, 13 *TaEXO70F* members were identified and *EXO70F1-V*, *EXO70F2-V* and *EXO70F3-V* were cloned from *H. villosa*. Expression studies have revealed that 11/13 common wheat varieties were induced by stress treatment (Figure 5), and three *EXO70-V* genes showed clearly diverse expression patterns, of which *EXO70F1-V* was only induced by SA, *EXO70F2-V* in response to drought, H2O<sup>2</sup> and SA at an early stage, and *EXO70F3-V* in response to *Bgt* treatment at a late stage (48 h) (Figure 6). Research shows that both SA and H2O<sup>2</sup> function as a key regulator against pathogens and stress tolerance [72–74]. Thus, we can infer the function of *EXO70F* genes not only in plant defense responses but also in abiotic stress.

In *N. benthamiana*, *EXO70D* and *EXO70G* mainly affect the size of the leaf [6]. In wheat, *TaEXO70D2-7BS* and *TaEXO70G1/G2* (with their homeoalleles) were induced by *Pst* and *Bgt* treatments. *EXO70D1-V* was upregulated by *Bgt* and MeJA treatments, *EXO70G1-V* had an increased expression level under phytohormones and H2O<sup>2</sup> treatments, while *EXO70G2-V* was induced by drought and SA. MeJA is important for regulating the growth of plants and promotes plant resistance of various stresses [75]. This might suggest that *EXO70D1-V* plays an important role in plant growth and defense responses *EXO70G* are multifunctional. Of 22 *EXO70I* genes from common wheat, only five genes had distinct inducible expression. For instance, *TaEXO70I6-7DL* and *TaEXO70I8-4BL* were upregulated by drought and heat, respectively. *EXO70I1-V* was induced by ET and SA and maintained a high expression level. ET and SA regulate many diverse metabolic and developmental processes in plants, such as seed germination, abiotic stress response and pathogen defense [76,77]. Thus, we hypothesize that *EXO70I1-V* might play a vital role in growth or against multiple stresses. *EXO70* genes provide diverse expression patterns in different tissues and stresses, implying that *EXO70* genes may play an essential role in plant adaptation to a complicated and changeable environment.
