Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer Transcription Activator-Like effectors (dTALes), which are synthetic type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce two independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the two dTALes. Nine of the ten genes known to be involved in cuticular wax biosynthesis were up-regulated by at least one of the two dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

1Rigorous exploration and dissection of potential actions and effects of long noncoding RNA 2 (lncRNA) in animals remain challenging. Here using multiple knockout mouse models and single-alleles of Hand2as in mouse (Han et al., 2018). Full-length deletion of the entire Hand2as sequence 1 (Hand2as F/F KO) led to dysregulated cardiac gene expression programs, ventricular septal defects 2 and heart hypoplasia and perinatal death, reminiscent of congenital heart diseases. A short distal 3 deletion at the 3' end of the Hand2as locus (Hand2as D/D KO) caused severe contraction defects in 4 adult heart that progressively worsened with increasing age. In comparison, short deletion of the 5' 5 promoter and exons of Hand2as (Hand2as P/P KO) effectively diminished Hand2as expression, but 6 failed to produce discernable heart phenotypes in either embryos or adults. These results indicate that 7 the Hand2as DNA locus, rather than its transcription/transcripts, primarily controls heart 8 development and function. To our surprise, cardiac expression of HAND2 was sustained in all three 9 Hand2as KO mouse models we generated, in sharp contrast to the abolished expression of HAND2 10 in the Uph/Hand2as polyA KI embryos (Anderson et al., 2016). Importantly, single-cell 11 transcriptomic analysis revealed subtle yet prevalent upregulation of HAND2 and concordant global 12 gene expression changes in subsets of cardiac cells of Hand2as F/F embryos lacking the entire Hand2as 13 DNA sequence. Altogether, these results illustrate a fine-tuning yet critical role for the lncRNA 14 Hand2as locus in restricting the precise, spatial expression of HAND2, through which Hand2as 15 modulates cardiac lineage development and heart function. This study reveals the unexpected 16 complexity of lncRNA function in vivo, and also emphasize the usage of complementary genetic and 17 single-cell approaches to delineate the primary molecular effects and elucidate physiological 18 functions of an lncRNA in animals.