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1. Structural variation affecting DNA backbone interactions underlies adaptation of B3 DNA binding domains to constraints imposed by protein architecture

   https://doi.org/10.1093/nar/gkab257  

   Jia, Haiyan; Suzuki, Masaharu; McCarty, Donald R (April 2021, Nucleic Acids Research)

   null (Ed.)

   Abstract Functional and architectural diversification of transcription factor families has played a central role in the independent evolution of complex development in plants and animals. Here, we investigate the role of architectural constraints on evolution of B3 DNA binding domains that regulate plant embryogenesis. B3 domains of ABI3, FUS3, LEC2 and VAL1 proteins recognize the same cis-element. Complex architectures of ABI3 and VAL1 integrate cis-element recognition with other signals, whereas LEC2 and FUS3 have reduced architectures conducive to roles as pioneer activators. In yeast and plant in vivo assays, B3 domain functions correlate with architectural complexity of the parent transcription factor rather than phylogenetic relatedness. In a complex architecture, attenuated ABI3-B3 and VAL1-B3 activities enable integration of cis-element recognition with hormone signaling, whereas hyper-active LEC2-B3 and FUS3-B3 over-ride hormonal control. Three clade-specific amino acid substitutions (β4-triad) implicated in interactions with the DNA backbone account for divergence of LEC2-B3 and ABI3-B3. We find a striking correlation between differences in in vitro DNA binding affinity and in vivo activities of B3 domains in plants and yeast. Our results highlight the role of DNA backbone interactions that preserve DNA sequence specificity in adaptation of B3 domains to functional constraints associated with domain architecture. 

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2. Comparative Transcriptomics of Non-Embryogenic and Embryogenic Callus in Semi-Recalcitrant and Non-Recalcitrant Upland Cotton Lines

   https://doi.org/10.3390/plants10091775  

   Kumar, Sonika; Ruggles, Ashleigh; Logan, Sam; Mazarakis, Alora; Tyson, Thomas; Bates, Matthew; Grosse, Clayton; Reed, David; Li, Zhigang; Grimwood, Jane; et al (September 2021, Plants)

   Somatic embryogenesis-mediated plant regeneration is essential for the genetic manipulation of agronomically important traits in upland cotton. Genotype specific recalcitrance to regeneration is a primary challenge in deploying genome editing and incorporating useful transgenes into elite cotton germplasm. In this study, transcriptomes of a semi-recalcitrant cotton (Gossypium hirsutum L.) genotype ‘Coker312’ were analyzed at two critical stages of somatic embryogenesis that include non-embryogenic callus (NEC) and embryogenic callus (EC) cells, and the results were compared to a non-recalcitrant genotype ‘Jin668’. We discovered 305 differentially expressed genes in Coker312, whereas, in Jin668, about 6-fold more genes (2155) were differentially expressed. A total of 154 differentially expressed genes were common between the two genotypes. Gene enrichment analysis of the upregulated genes identified functional categories, such as lipid transport, embryo development, regulation of transcription, sugar transport, and vitamin biosynthesis, among others. In Coker312 EC cells, five major transcription factors were highly upregulated: LEAFY COTYLEDON 1 (LEC1), WUS-related homeobox 5 (WOX5), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and WRKY2. In Jin668, LEC1, BABY BOOM (BBM), FUS3, and AGAMOUS-LIKE15 (AGL15) were highly expressed in EC cells. We also found that gene expression of these embryogenesis genes was typically higher in Jin668 when compared to Coker312. We conclude that significant differences in the expression of the above genes between Coker312 and Jin668 may be a critical factor affecting the regenerative ability of these genotypes. 

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3. Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control

   https://doi.org/10.1111/mpp.13361  

   McRae, Amanda G; Taneja, Jyoti; Yee, Kathleen; Shi, Xinyi; Haridas, Sajeet; LaButti, Kurt; Singan, Vasanth; Grigoriev, Igor V; Wildermuth, Mary C (September 2023, Molecular Plant Pathology)

   Abstract Spray‐induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double‐stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole‐fungicide targetcytochrome P45051 (CYP51) in theGolovinomyces orontii–Arabidopsis thalianapathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation:apoptosis‐antagonizing transcription factorin essential cellular metabolism and stress response; lipid catabolism geneslipase a,lipase 1, andacetyl‐CoA oxidasein energy production;and genes involved in manipulation of the plant host via abscisic acid metabolism (9‐cis‐epoxycarotenoid dioxygenase,xanthoxin dehydrogenase, and a putativeabscisic acid G‐protein coupled receptor) and secretion of the effector protein,effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for theErysiphe necator–Vitis viniferasystem and tested six successful targets identified using theG. orontii–A. thalianasystem. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in theG. orontii–A. thalianapathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control. 

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4. A RanBP2-type zinc finger protein functions in intron splicing in Arabidopsis mitochondria and is involved in the biogenesis of respiratory complex I

   https://doi.org/10.1093/nar/gkab066  

   Bentolila, Stéphane; Gipson, Andrew B; Kehl, Alexander J; Hamm, Lauren N; Hayes, Michael L; Mulligan, R Michael; Hanson, Maureen R (February 2021, Nucleic Acids Research)

   Abstract The RanBP2 zinc finger (Znf) domain is a prevalent domain that mediates protein interaction and RNA binding. In Arabidopsis, a clade of four RanBP2 Znf-containing proteins, named the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to either the mitochondria or the plastids. Previously we reported that OZ1 is absolutely required for the editing of 14 sites in chloroplasts. We now have investigated the function of OZ2, whose null mutation is embryo lethal. We rescued the null mutant by expressing wild-type OZ2 under the control of the seed-specific ABSCISIC ACID-INSENSITIVE3 (ABI3) promoter. Rescued mutant plants exhibit severely delayed development and a distinctive morphological phenotype. Genetic and biochemical analyses demonstrated that OZ2 promotes the splicing of transcripts of several mitochondrial nad genes and rps3. The splicing defect of nad transcripts results in the destabilization of complex I, which in turn affects the respiratory ability of oz2 mutants, turning on the alternative respiratory pathway, and impacting the plant development. Protein-protein interaction assays demonstrated binding of OZ2 to several known mitochondrial splicing factors targeting the same splicing events. These findings extend the known functional repertoire of the RanBP2 zinc finger domain in nuclear splicing to include plant organelle splicing. 

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5. Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

   https://doi.org/10.1038/s41467-023-38096-2  

   Li, Miaomiao; Yao, Tao; Lin, Wanru; Hinckley, Will E.; Galli, Mary; Muchero, Wellington; Gallavotti, Andrea; Chen, Jin-Gui; Huang, Shao-shan Carol (May 2023, Nature Communications)

   Abstract Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo-versusheterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers inArabidopsisand show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation. 

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