## 3 Materials and Methods
### Analysis of the Binding of ANAC074 to the Truncated Promoters  
The sequences of MybSt1, NRS1, and NRS2 were searched using the Patmatch program on Tair [33] to find the genes that were putatively regulated by ANAC074. 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 having 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 pHIS2 harboring the truncated promoter of _AT4G17500_ (without NACRS, NRS2 or MybSt1) containing NRS1 \"TAGTT\" (pHIS2-NRS1p+), without \"TAGTT\" (pHIS2-NRS1p-) or with NRS1 complete mutant \"GGGGG\" (pHIS2-NRS1 pm) driving _HIS3_ gene, were respectively generated as reporter vectors (see Supplemental Table S1 for primers used). The pHIS2 harboring the truncated promoter of _AT1G74930_ (without NACRS, NRS1, or MybSt1) containing NRS2 \"GAATC\" (pHIS2-NRS2p+), without \"GAATC\" (pHIS2-NRS2p-) or NRS2 complete mutant \"TCCGA\" (pHIS2-NRS2 pm) driving _HIS3_ gene were respectively generated as reporter vectors (see Supplemental Table S1 for primers used). Similarly, the pHIS2 harboring the truncated promoter of _AT4G11280_ (without NACRS, NRS1, or NRS2) containing MybSt1 \"GGATA\" (pHIS2-MYBp+), without \"GGATA\" (pHIS2-MYBp-) or MybSt1 complete mutant \"TTCGC\" driving _HIS3_ gene were also respectively generated as reporter vectors (see Supplemental Table S1 for primers used). The interactions of these sequences with the ANAC074 were studied using Y1H analysis [23; 24].  
To further verify the Y1H results, transient expression assays in vivo were performed. The reporter vectors harboring the truncated promoter of _AT4G17500_ (same as used in Y1H) containing NRS1 \"TAGTT\" (pCAM-NRS1p+), without NRS1 (pCAM-NRS1p-) or NRS1 complete mutant \"GGGGG\" (pCAM-NRS1 pm), were respectively generated (see Supplemental Table S1 for primers used). Similarly, the truncated promoter of _AT1G74930_ (same as used in Y1H), which contained NRS2 \"GAATC\" (pCAM-NRS2p+), without \"GAATC\" (pCAM-NRS2p-) or NRS2 completely mutant \"TCCGA\" (pCAM-NRS2 pm) were respectively fused to the minimal 35S promoter (\(-\)46 to \(+\)1) to substitute 35S promoter to drive _GUS_ as reporter vectors (see Supplemental Table S1 for primers used). The reporter vectors harboring the truncated promoter of _AT4G11280_ (same as used in Y1H) containing MybSt1 motif \"GGATA\" (pCAM-MYBp+), without \"GGATA\" (pCAM-MYBp-) or MybSt1 complete mutant motif \"TTCGC\" (pCAM-MYBpm) were also respectively generated. (See Supplemental Table S1 for primers used).  
These reporter constructs were respectively co-transformed with effector (pROKII-ANAC074) into tobacco leaves using the particle bombardment method (Bio-Rad, Hercules, CA, USA), respectively. GUS activity levels were determined according to the method of [PERSON] [46]. Three independent experiments were performed. The transformation of single reporter plasmid and the empty effector vector were used as a negative control. The empty pCAMBIA1301 was transformed and used as positive control.