## 2 Results and Discussion
### Binding of Two Novel Motifs and MybSt1 Motif to ANAC074
The effector was constructed by cloning the ORF of ANAC074 into pROKII under 35 SCAMV promoter. Three tandem copies of NRS1, NRS2, and MybSt1 (black shading) or their complete mutant types NRS1-M3, NRS2-M3, and MYB-M3 (grey shading) were respectively fused to the minimal 35S promoter for driving _GUS_ as reporter vectors. (**C**) Transient co-transformation of effector and reporter constructs into tobacco leaves to study the specific bindings of NRS1, NRS2, and MybSt1 to ANAC074. GUS activity was determined to study the binding affinity between the test DNA sequence and ANAC074, and the combinations between effector and reporter construct are shown in the left panel. The empty pCAMBIA1301 was as positive control. Co-transformed empty pROKII with pCAM-NRS1, pCAM-NRS2 or pCAM-MYBB were as negative controls. Data represent mean values of three independent experiments, and bars indicate standard deviation (SD). Asterisks indicate a significant difference compared with the mutation (Student’s test; \({}^{*}\)\(p<\)0.05). Furthermore, the MybSt1 motif was found previously to be bound by MYB transcription factors MybStl that encode a novel Myb-related protein from potato [26]. MybStl requires a GGATA-containing sequence for high-affinity binding. GGATG possesses a 20-30 times lower binding affinity than GGATA, which suggested the fifth \"A\" in the GGATA sequences is important for MybStl recognition [26]. Our results showed that ANAC074 could recognize the GGATA (MybStl), but failed to interact with the mutated motifs \(\underline{TT}\)ATA (M1), \(\underline{GT}\)CGA (M2) and \(\underline{TT}\)CGC (M3) (Figure 2A), indicating that the bases \"GG\" and \"GAT\" are also essential to ANAC074 to bind to MybSt1 motif. MYB transcription factors perform diverse critical functions in plants, including secondary metabolism [27], development [28], hormonal signaling [29], and biotic stress response [30]. Increasing evidence in model plants show that MYB genes are also involved in tolerance to abiotic stress [31; 32]. The binding of ANAC074 to the MybSt1 motif suggested that it might have similar functions to the MYB TFs. NAC proteins may share regulatory networks with MYB proteins by binding to the MybSt1 binding site. The two TFs can be competitive or cooperative in the same signaling pathway. ANAC074 Can Bind the Promoters of AT4G17500, AT1G74930 and AT4G11280 via the NRS1, NRS2 or MybSt1 Element  
The MybSt1, NRS1, and NRS2 were identified using the TF-centered Y1H system [23; 24]. However, to eliminate artifacts, we further searched the NRS1, NRS2, and MybSt1 sequences to find the genes that were putatively regulated by ANAC074 from _Arabidopsis_ using the Patmatch program on Tair [33]. The criteria for promoter selection were the promoter regions (0 to \(-\)1500 bp) of genes containing fewer kinds of the motifs of MybSt1, NRS1, and NRS2, but have more copies of the contained motif. We found a total of 33 genes that met the criteria. Three of them (_AT4G17500, AT1G74930_ and _AT4G11280_) happen to be the target genes of ANAC069 [22]. We speculated that these three genes are probably also the target genes of ANAC074. Therefore, these three genes were selected for the promoter mutagenesis. The promoter analysis showed that NRS1 was present in the promoter regions of _ATERF-1 (AT4G17500)_, NRS2 was present in the promoter regions of _ORA47 (AT1G74930)_, and MybSt1 element was included in _ATACS6 (AT4G11280)_ (Figure 3A).