Biology subjects

Reddy, A. S. N.

Publications and source records attributed to Reddy, A. S. N..

5 recordsLinked to original sources

bioRxiv2023

DNA binding activity of CAMTA3 is essential for its function

Calmodulin-binding transcription activators (CAMTAs), a small family of highly conserved transcription factors, function in calcium-mediated signaling pathways. Of the six CAMTAs in Arabidopsis, CAMTA3 (also referred to as SR1) regulates diverse biotic and abiotic stress responses. A recent study has shown that CAMTA3 is a guardee of NLR ((nucleotide-binding, leucine-rich repeat domain) immune receptors in modulating plant immunity, thereby suggesting that CAMTA3 transcriptional activity is not necessary for its function. Here, we show that the DNA-binding activity of CAMTA3 is essential for its role in mediating plant immune responses. Analysis of the DNA binding (CG-1) domain of CAMTAs in plants and animals showed strong conservation of several amino acids. We mutated several conserved amino acids in the CG-1 domain to investigate their role in CAMTA3 function. Electrophoretic mobility shift assays using these mutants with a promoter of its target gene identified critical amino acid residues necessary for DNA binding activity. Furthermore, transient assays showed that these residues are essential for the CAMTA3 function in activating the RSRE (Rapid Stress Response Element)-driven reporter gene expression. In line with this, transgenic lines expressing the CG-1 mutants of CAMTA3 in the camta3 mutant failed to rescue the mutant phenotype and restore the expression of CAMTA3 downstream target genes. Collectively, our results provide biochemical and genetic evidence that the transcriptional activity of CAMTA3 is indispensable for its function.

plant biology↗

bioRxiv2021

Evidence for the role of transcription factors in the co-transcriptional regulation of intron retention

Alternative splicing is a widespread regulatory phenomenon that enables a single gene to produce multiple transcripts. Among the different types of alternative splicing, intron retention is one of the least explored despite its high prevalence in both plants and animals. The recent discovery that the majority of splicing is co-transcriptional has led to the finding that chromatin state affects alternative splicing. Therefore it is plausible that transcription factors can regulate splicing outcomes. We provide evidence for this hypothesis by studying regions of open chromatin in retained and excised introns. Using deep learning models designed to distinguish between regions of open chromatin in retained introns and non-retained introns, we identified motifs enriched in IR events with significant hits to known human transcription factors. Our model predicts that the majority of transcription factors that affect intron retention come from the zinc finger family. We demonstrate the validity of these predictions using ChIP-seq data for multiple zinc finger transcription factors and find strong over-representation for their peaks in intron retention events. AvailabilitySource code available at https://github.com/fahadahaf/chromir

genomics↗

bioRxiv2021

A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation or incomplete cDNA synthesis. ResultsWe present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 160k transcripts - twice that of the best current Arabidopsis transcriptome and including over 1,500 novel genes. 79% of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis- match profiles around splice junctions provided a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identified high confidence transcription start/end sites and removed fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provided higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start and polyadenylation site usage. ConclusionsAtRTD3 is the most comprehensive Arabidopsis transcriptome currently available. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.

plant biology↗

bioRxiv2021

An in-frame deletion mutation in the degron tail of auxin co-receptor IAA2 confers resistance to the herbicide 2,4-D in Sisymbrium orientale

The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides mimic the effects of IAA by inducing strong auxinic signaling responses in plants. Synthetic auxins are crucial herbicides in agriculture, made more important by the recent introduction of transgenic synthetic auxin resistant soybean and cotton. Currently, 41 weed species have evolved resistance to synthetic auxin herbicides and, in all but one case, the molecular basis of these resistance mechanisms is unknown. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and-susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing 9 amino acids in the degron tail (DT) of the auxin co-receptor Aux/IAA2 (SoIAA2). The deletion allele co-segregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This novel mechanism of synthetic auxin herbicide resistance assigns a new in planta function to the DT region of this Aux/IAA co-receptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin resistant crops using gene editing.

plant biology↗

bioRxiv2019

Overexpression of the Arabidopsis thaumatin-like protein 1 in transgenic potato plants enhances resistance against early and late blights

The Arabidopsis thaumatin-like protein 1 (ATLP1), which belongs to the PR5 family of pathogenesis-related proteins, is induced by pathogen attacks and by systemic acquired resistance (SAR)-inducing compounds. To test whether the overexpression of ATLP1 will enhance fungal resistance in transgenic plants, the cDNA of this gene under the control of a constitutive promoter was transferred into Solanum tuberosum Cv. Desiree. The expression of the introduced gene was confirmed by Northern and Western blot analyses. Western blot analysis performed with intercellular fluid (ICF) showed that ATLP1 is secreted to apoplast. Several independent transgenic lines with high-level expression of the ATLP1 were assayed for resistance against early blight (Alternaria solani) and late blight (Phytophthora infestans). The rate of Alternaria lesions development was significantly reduced in the ATLP1 transgenic lines as compared to a control line. Percent reductions in area under the disease progress curve (AUDPC) values for the ATLP1 transgenic lines as compared to control line ranged between 52 and 82%. In response to P. infestans, infection efficiency (IE), lesion size (LS) and sporulation capacity (SC) were significantly reduced in ATLP1 transgenic lines as compared to a control line. On the average, IE, LS and SC were reduced by 18, 22 and 20%, respectively, for all transgenic lines with a maximum reduction of 25% in IE, 25% in LS and 32% in SC. Resistance assays against P. infestans using whole plants showed 40-70% reduction in symptoms as compared to control. These results suggest that constitutive expression of a heterologous ATLP in potato confers enhanced resistance against early and late blights.

plant biology↗