Biology subjects

Siddiqi, I.

Publications and source records attributed to Siddiqi, I..

7 recordsLinked to original sources

bioRxiv2025

CDM1 is required for meiotic progression and genome integrity during male meiosis in Arabidopsis

Microsporogenesis involves the integration of meiosis with the developmental program leading to formation of microspores from meiocytes. The CALLOSE DEFICIENT MICROSPORE 1 (CDM1) gene of Arabidopsis has been previously characterized as being required for callose metabolism and its loss results in defects in microspore wall development, degeneration of meiotic products, and male sterility. We report here that CDM1 is required for normal meiosis I progression, meiotic chromosome organization, and maintenance of DNA integrity. CDM1 is part of the ATM dependent DNA damage response in dividing vegetative tissues. The cdm1 mutant shows defects in meiotic chromosome condensation, arrest at metaphase I, susceptibility to chromosome breakage in rDNA regions, defects in positioning of the products of meiosis, and increased expression of recombination related genes. However, fidelity of chromosome segregation is not affected. Furthermore, the meiotic phenotype of cdm1 is independent of SPO11 induced meiotic double strand breaks. CDM1 protein displays granular localization in meiosis throughout the cytoplasm that resembles that of mRNP granules. The results show that CDM1 is required for maintenance of proper chromosome organization and DNA integrity in the course of male meiosis. The chromosomal defects in cdm1 may originate from regions of defective or incomplete replication. HighlightCDM1 expression is DNA damage inducible in Arabidopsis mitotic cells. CDM1 is required for genome integrity in male meiosis independent of SPO11-induced DNA breaks, suggesting a role in regulation of premeiotic replication repair.

plant biology↗

bioRxiv2024

Sporophyte Directed Gametogenesis via the Ubiquitin Proteasome System

Plants alternate between diploid sporophyte and haploid gametophyte generations. In mosses which retain features of ancestral land plants, the gametophyte is dominant and has an independent existence. However, in flowering plants the gametophyte has undergone evolutionary reduction to just a few cells enclosed within the sporophyte. The gametophyte is thought to retain genetic control of its development even after reduction. Here we demonstrate that male gametophyte development in Arabidopsis, long considered to be autonomous, is also under genetic control of the sporophyte via a repressive mechanism involving large-scale regulation of protein turnover. We identify an Arabidopsis gene SHUKR as an inhibitor of male gametogenesis. SHUKR is unrelated to proteins of known function and acts sporophytically in meiosis to control gametophyte development by negatively regulating expression of a large set of ubiquitination genes specific to post-meiotic gametogenesis. This control is late-emerging as SHUKR homologs are found only in eudicots. We show that SHUKR is rapidly evolving under positive selection suggesting that variation in control of protein turnover during male gametogenesis has played an important role in evolution within eudicots.

plant biology↗

bioRxiv2024

Improved Validation of Protein Interactions usingBicistronic BiFC (Bi2FC)

Refolding based Bimolecular Fluorescence Complementation (BiFC) has emerged as an important in vivo technique to identify protein interactions. Significant improvements have been made to enhance the detection capacities of BiFC, however less attention has been paid to the detection of expression levels of proteins. Here we demonstrate development and validation of an improved method to identify protein interactions that incorporates an expression control based on bicistronic expression of the protein of interest and a fluorescent protein separated by a self-cleaving peptide. This method gives robust identification of positive interactions and more reliably identifies absence of interactions. We also show an earlier identified non-interacting pair in yeast two-hybrid (Y2H) to be interacting in vivo. BackgroundBimolecular Fluorescence Complementation (BiFC) is a well-utilized method to detect protein interactions in vivo (Kodama and Hu 2012). It is dependent on the refolding of a fluorescent protein split into two parts. The proximity between the two parts leads to functional refolding of the protein and a corresponding increase in the fluorescence intensity. Proteins of interest can be tagged to either part of the split fluorescent protein and be expressed together in a single cell. The physical proximity provided due to interactions between the proteins brings the two parts of the split fluorescent protein closer leading to the formation of a functional fluorescent complex. BiFC has at times been used as the sole technique to identify protein interactions in vivo or else as a step following identification of candidate positives by yeast two-hybrid (Y2H). In order to identify the interactions reliably it is also important to validate the gene expression status of BiFC elements (Kudla and Bock 2016). However, systems to visualize the interactions and expression status together are not available for plants. ResultsWe generated and validated a vector system, called Bi2FC (Bicistronic BiFC) vector system for verifying protein interactions in plants. Bicistronic expression of proteins of interest fused with the two different BiFC components and a fluorescent protein, separated by a self-cleaving peptide identified the expression status of the fusion, while simultaneously identifying positive and negative interactions. As a test, we identified interaction between MPK8 and GRF4 using our system, which were previously identified to not show interaction via Y2H. ConclusionWe described an improved BiFC method to current BiFC to identify the expression of individual proteins tagged with split fluorescent protein as well as to monitor their interaction in a plant cell. This method incorporates additional controls that express only the split fragments together with a vector control, while also providing a set of validated positive and negative controls. Using our method, we also show a previously identified non-interacting pair in yeast two-hybrid (Y2H) to be interacting, emphasizing the need for improved detection and validations.

molecular biology↗

bioRxiv2023

Archaeal origin translation proofreader imparts multialdehyde stress tolerance to land plants

Aldehydes, being an integral part of carbon metabolism, energy generation and signalling pathways, are ingrained in plant physiology. Land plants have developed intricate metabolic pathways which involve production of reactive aldehydes and its detoxification to survive harsh terrestrial environments. Here, we show that physiologically produced aldehydes i.e., formaldehyde and methylglyoxal in addition to acetaldehyde, generate adducts with aminoacyl-tRNAs, a substrate for protein synthesis. Plants are unique in possessing two distinct chiral proofreading systems, D-aminoacyl-tRNA deacylase1 (DTD1) and DTD2, of bacterial and archaeal origins, respectively. Extensive biochemical analysis revealed that only archaeal DTD2 can remove the stable D-aminoacyl adducts on tRNA thereby shielding archaea and plants from these system-generated aldehydes. Using Arabidopsis as a model system, we have shown that the loss of DTD2 gene renders plants susceptible to these toxic aldehydes as they generate stable alkyl modification on D-aminoacyl-tRNAs, which are recycled only by DTD2. Bioinformatic analysis identifies the expansion of aldehyde metabolising repertoire in land plant ancestors which strongly correlates with the recruitment of archaeal DTD2. Finally, we demonstrate that the overexpression of DTD2 offers better protection against aldehydes than in wild-type Arabidopsis highlighting its role as a multi-aldehyde detoxifier that can be explored as a transgenic crop development strategy.

plant biology↗

bioRxiv2022

The FANCC-FANCE-FANCF complex is evolutionarily conserved and regulates meiotic recombination

At meiosis, programmed meiotic DNA double-strand breaks are repaired via homologous recombination, resulting in crossovers (COs). From a large excess of DNA double-strand breaks that are formed, only a small proportion gets converted into COs because of active mechanisms that restrict CO formation. The Fanconi anemia (FA) complex proteins AtFANCM, MHF1, and MHF2 were previously identified in a genetic screen as anti-CO factors that function during meiosis in Arabidopsis thaliana. Here, pursuing the same screen, we identify FANCC as a new anti-CO gene. FANCC was previously only identified in mammals because of low primary sequence conservation. We show that FANCC, and its physical interaction with FANCE-FANCF, is conserved from vertebrates to plants. Further, we show that FANCC, together with its subcomplex partners FANCE and FANCF, regulates meiotic recombination. Mutations of any of these three genes partially rescues CO-defective mutants, which is particularly marked in female meiosis. Functional loss of FANCC, FANCE, or FANCF results in synthetic meiotic catastrophe with the pro-CO factor MUS81. This work reveals that FANCC is conserved outside mammals and has an anti-CO role during meiosis together with FANCE and FANCF.

genetics↗

bioRxiv2020

An Inexpensive RT-PCR Endpoint Diagnostic Assay for SARS-CoV-2 Using Nested PCR: Direct Assessment of Detection Efficiency of RT-qPCR Tests and Suitability for Surveillance

With a view to extending testing capabilities for the ongoing SARS-CoV-2 pandemic we have developed a test that lowers cost and does not require real time quantitative reverse transcription polymerase chain reaction (RT-qPCR). We developed a reverse transcription nested PCR endpoint assay (RT-nPCR) and showed that RT-nPCR has comparable performance to the standard RT-qPCR test. In the course of comparing the results of both tests, we found that the standard RT-qPCR test can have low detection efficiency (less than 50%) in a real testing scenario which may be only partly explained by low viral representation in many samples. This finding points to the importance of directly monitoring detection efficiency in test environments. We also suggest measures that would improve detection efficiency.

molecular biology↗

bioRxiv2019

The TARANI/ UBIQUITIN SPECIFIC PROTEASE 14 destabilizes the AUX/IAA transcriptional repressors and regulates auxin response in Arabidopsis thaliana

Auxin response is regulated by a group of AUX/IAA transcriptional inhibitors that suppress auxin signaling in the absence of the hormone. While the degradation of these proteins upon auxin signaling has been well studied, the molecular control of their rapid turn-over is not clearly understood. Here, we report that the TARANI/ UBIQUITIN PROTEASE 14 protein in Arabidopsis thaliana (Arabidopsis) is required for AUX/IAA degradation. The tni mutation was originally identified in a forward genetic screen to isolate mutants with altered leaf shape. Detailed phenotypic analysis revealed that tni displays pleiotropic phenotypic alterations that resemble auxin-related defects. The activity of auxin responsive reporters DR5::GUS, DR5::nYFP and IAA2::GUS was reduced in tni organs, implying that TNI is required for normal auxin response. Genetic interaction studies suggested that TNI acts along with TIR1, ARF7, AUX1 and PIN1 - molecules involved in auxin signaling or transport. A map-based cloning approach combined with next-generation sequencing identified TNI as UBIQUITIN SPECIFIC PROTEASE14 which is involved in ubiquitin recycling. In tni, the mutant primary transcript is spliced inefficiently, which is predicted to produce an aberrant protein product in addition to the normal protein, where a polypeptide corresponding to the 3rd intron in inserted in-frame within the Zn-finger domain of UBP14. The tni plants accumulated poly-ubiquitin chains and excess poly-ubiquitinated proteins due to reduced TNI activity. Improper ubiquitin recycling affected the degradation of DII:VENUS, IAA18:GUS and HS::AXR3-NT:GUS, resulting in their stabilization in the tni mutant. Thus, our study identified a function for TNI/UBP14 in regulating auxin response through ubiquitin recycling.

plant biology↗