Biology subjects

Arazi, T.

Publications and source records attributed to Arazi, T..

5 recordsLinked to original sources

bioRxiv2024

Knockout of the tomato HAIRY MERISTEM 4 alters phloem-characteristics and impairs development

The HAIRY MERISTEM (HAM) gene family encodes Type I and II GRAS domain transcription factors in plants. Type II HAMs, predominantly expressed in meristems and regulated by microRNA171, are essential for maintaining undifferentiated meristems, a role conserved across various species. Conversely, the functions of Type I HAMs have been less characterized. In this study, we investigated the role of SlHAM4, a Type I HAM in tomato. Using publicly available expression data and a GUS reporter gene driven by the native SlHAM4 promoter, we determined that SlHAM4 is predominantly expressed in phloem tissues. CRISPR-induced SlHAM4 loss-of- function mutations (slham4CR) resulted in a range of shoot and fruit abnormalities, which were fully reversed by reintroducing SlHAM4 under its native promoter in the mutant background. Mutant abnormalities included increased anthocyanin pigmentation in the leaf and sepal primordia, reminiscent of the phenotypes observed in certain Arabidopsis mutants with compromised phloem, and development of simpler leaves, which was associated with reduction in external phloem area in the leaf rachis. In addition, slham4CR plants produced significantly smaller fruits of which a fraction of them exhibited catface-like scars, attributed to tears which occurred in the pericarp of mutant ovaries following fruit set. Transcriptome analysis of the wild-type looking mutant ovaries at anthesis revealed specific downregulation of genes implicated in phloem development and functions, in particular those expressed in companion cells (CC). We propose that SlHAM4 is necessary for proper phloem function in part by regulating the expression of a suite of CCs genes that encode essential phloem proteins. One-sentence summarySlHAM4 is predominantly expressed in the phloem and its knockout alters phloem- characteristics and impair development highlighting its requirement for proper phloem functionality.

plant biology↗

bioRxiv2024

Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase

Epigenetic variation is mediated by epigenetic marks such as DNA methylation occurring in all cytosine contexts in plants. CG methylation plays a critical role in silencing transposable elements and regulating gene expression. The establishment of CG methylation occurs via the RNA-directed DNA methylation pathway and CG methylation maintenance relies on METHYLTRANSFERASE1, the homologue of the mammalian DNMT1. Here, we examined the capacity to stably alter the tomato genome methylome by a bacterial CG-specific M.SssI methyltransferase expressed through the LhG4/pOP transactivation system. Methylome analysis of M.SssI expressing plants revealed that their euchromatic genome regions are specifically hypermethylated in the CG context, and so are most of their genes. However, changes in gene expression were observed only with a set of genes exhibiting a greater susceptibility to CG hypermethylation near their transcription start site. Unlike gene rich genomic regions, our analysis revealed that heterochromatic regions are slightly hypomethylated at CGs only. Notably, some M.SssI-induced hypermethylation persisted even without the methylase or transgenes, indicating inheritable epigenetic modification. Collectively our findings suggest that heterologous expression of M.SssI can create new inherited epigenetic variations and changes in the methylation profiles on a genome wide scale. This open avenues for the conception of epigenetic recombinant inbred line populations with the potential to unveil agriculturally valuable tomato epialleles.

plant biology↗

bioRxiv2023

A virally encoded high resolution screen of cytomegalovirus host dependencies

Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins role along the infection cycle, providing a roadmap for in-depth dissection of host-herpesvirus interactions.

microbiology↗

bioRxiv2022

The molecular principles governing HCMV infection outcome

Infection with Human cytomegalovirus (HCMV) can result in either productive or non-productive infection, the latter potentially leading to establishment of latency, but the molecular factors that dictate these different infection outcomes are elusive. Macrophages are known targets of HCMV and considered to be permissive for productive infection, while monocytes, their precursors, are latently infected. Here we reveal that infection of macrophages is more complex than previously appreciated and can result in either productive or non-productive infection. By analyzing the progression of HCMV infection in monocytes and macrophages using single cell transcriptomics, we uncover that the level of viral gene expression, and specifically the expression of the major immediate early proteins, IE1 and IE2, is the principal barrier for establishing productive infection. On the cellular side, we reveal that the cell intrinsic levels of interferon stimulated genes (ISG), but not their induction, is a main determinant of infection outcome and that intrinsic ISG levels are downregulated with monocyte differentiation, partially explaining why macrophages are more susceptible to productive HCMV infection. We further show that, compared to monocytes, non-productive macrophages maintain higher levels of viral transcripts and are able to reactivate, raising the possibility that they may serve as latency reservoirs. Overall, by harnessing the tractable system of monocyte differentiation we decipher underlying principles that control HCMV infection outcome, and propose macrophages as a potential HCMV reservoir in tissues.

microbiology↗

bioRxiv2021

CLASS-II KNOX genes coordinate spatial and temporal patterns of the tomato ripening

Ripening is a complex developmental change of a mature organ, the fruit. In plants like a tomato, it involves softening, pigmentation, and biosynthesis of metabolites beneficial for the human diet. Examination of the transcriptional changes towards ripening suggests that redundant uncharacterized factors may be involved in the coordination of the ripening switch. Previous studies have demonstrated that Arabidopsis CLASS-II KNOX genes play a significant role in controlling the maturation of siliques and their transition to senescence. Here we examined the combined role of all four tomato CLASS-II KNOX genes in the maturation and ripening of fleshy fruits using an artificial microRNA targeting them simultaneously. As expected, the knockdown plants (35S::amiR-TKN-CL-II) exhibited leaves with increased complexity, reminiscent of the leaf phenotype of plants overexpressing CLASS-I KNOX, which antagonize CLASS-II KNOX gene functions. The fruits of 35S::amiR-TKN-CL-II plants were notably smaller than the control. While their internal gel/placenta tissue softened and accumulated the typical pigmentation, the pericarp color break took place ten days later than control, and eventually, it turned yellow instead of red. Additionally, the pericarp of 35S::amiR-TKN-CL-II fruits remained significantly firmer than control even after three weeks of shelf storage. Strikingly, the 35S::amiR-TKN-CL-II fruits showed early ethylene release and respiration peak, but these were correlated only with liquefaction and pigmentation of the internal tissues. Our findings suggest that CLASS-II KNOX genes are required to coordinate the spatial and temporal patterns of tomato fruit ripening. One sentence summaryTomato CLASS-II KNOX genes play antagonistic roles in the regulation of ripening at the internal fruit domains and pericarp.

developmental biology↗