Hyperubiquitylation of DNA helicase RECQL4 by E3 ligase MITOL prevents mitochondrial entry and potentiates mitophagy.

Mutations in the DNA helicase RECQL4 lead to Rothmund-Thomson syndrome (RTS), a disorder characterized by mitochondrial dysfunctions, premature aging, and genomic instability. However, the mechanisms by which these mutations lead to pathology are unclear. Here we report that RECQL4 is ubiquitylated by a mitochondrial E3 ligase, MITOL, at two lysine residues (K1101, K1154) via K6 linkage. This ubiquitylation hampers the interaction of RECQL4 with mitochondrial importer Tom20, thereby restricting its own entry into mitochondria. We show the RECQL4 2K mutant (where both K1101 and K1154 are mutated) has increased entry into mitochondria and demonstrates enhanced mitochondrial DNA (mtDNA) replication. We observed that the three tested RTS patient mutants were unable to enter the mitochondria and showed decreased mtDNA replication. Furthermore, we found that RECQL4 in RTS patient mutants are hyperubiquitylated by MITOL and form insoluble aggregate-like structures on the outer mitochondrial surface. However, depletion of MITOL allows RECQL4 expressed in these RTS mutants to enter mitochondria and rescue mtDNA replication. Finally, we show increased accumulation of hyperubiquitylated RECQL4 outside the mitochondria leads to the cells being potentiated to increased mitophagy. Hence, we conclude regulating the turnover of RECQL4 by MITOL may have a therapeutic effect in patients with RTS.

OsRuvBL1a DNA helicase boost salinity and drought tolerance in transgenic indica rice raised by in planta transformation.

RuvBL, is a member of SF6 superfamily of helicases and is conserved among the various model systems. Recently, rice (Oryza sativa L.) homolog of RuvBL has been biochemically characterized for its ATPase and DNA helicase activities; however its involvement in stress has not been studied so far. Present investigation reports the detailed functional characterization of OsRuvBL under abiotic stresses through genetic engineering. An efficient Agrobacterium-mediated in planta transformation protocol was developed in indica rice to generate the transgenic lines and study was focused on optimization of factors to achieve maximum transformation efficiency. Overexpressing OsRuvBL1a transgenic lines showed enhanced tolerance under in vivo salinity stress as compared to WT plants. The physiological and biochemical analysis of the OsRuvBL1a transgenic lines showed better performance under salinity and drought stresses. Several stress responsive interacting partners of OsRuvBL1a were identified using Y2H system revealed to its role in stress tolerance. Functional mechanism for boosting stress tolerance by OsRuvBL1a has been proposed in this study. This integration of OsRuvBL1a gene in rice genome using in planta transformation method helped to achieve the abiotic stress resilient smart crop. This study is the first direct evidence to show the novel function of RuvBL in boosting abiotic stress tolerance in plants.

Protocol to detect in vitro and in cell ubiquitylation of mitochondrial DNA polymerase gamma by mitochondrial E3 ligase MITOL.

Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro. For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021).

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria.

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

Rice lectin receptor-like kinase provides salinity tolerance by ion homeostasis.

Sensing stress and activating the downstream signaling pathways is the imperative step for stress response. Lectin receptor-like kinase (LecRLK) is an important family that plays a key role in sensing stress conditions through lectin receptor and activates downstream signaling by kinase domain. We identified the role of OsLecRLK gene for salinity stress tolerance and hypothesized its role in Na+ extrusion from cell. OsLecRLK overexpression and downregulation (through artificial miRNA) transgenic lines were developed and its comparison with wild-type (WT) plants were performed overexpression transgenic lines showed better performance, whereas downregulation showed poor performance than WT. Lower accumulation of reactive oxygen species (ROS), malondialdehyde and toxic ion, and a higher level of proline, RWC, ROS scavengers in overexpression lines lead us to the above conclusion. Based on the relative expression of stress-responsive genes, ionic content and interactome protein, working model highlights the role of OsLecRLK in the extrusion of Na+ ion from the cell. This extrusion is facilitated by a higher expression of salt overly sensitive 1 (Na+ /K+ channel) in overexpression transgenic line. Altered expression of stress-responsive genes and changed biochemical and physiological properties of cell suggests an extensive reprogramming of the stress-responsive metabolic pathways by OsLecRLK under stress condition, which could be responsible for the salt tolerance capability.

Marker-free transgenic rice plant overexpressing pea LecRLK imparts salinity tolerance by inhibiting sodium accumulation.

KEY MESSAGE: PsLecRLK overexpression in rice provides tolerance against salinity stress and cause upregulation of SOS1 pathway genes, which are responsible for extrusion of excess Na+ ion under stress condition. Soil salinity is one of the most devastating factors threatening cultivable land. Rice is a major staple crop and immensely affected by soil salinity. The small genome size of rice relative to wheat and barley, together with its salt sensitivity, makes it an ideal candidate for studies on salt stress response caused by a particular gene. Under stress conditions crosstalk between organelles and cell to cell response is imperative. LecRLK is an important family, which plays a key role under stress conditions and regulates the physiology of the plant. Here we have functionally validated the PsLecRLK gene in rice for salinity stress tolerance and hypothesized the model for its working. Salt stress sensitive rice variety IR64 was used for developing marker-free transgenic with modified binary vector pCAMBIA1300 overexpressing PsLecRLK gene. Comparison of transgenic and wild-type (WT) plants showed better physiological and biochemical results in transgenic lines with a low level of ROS, MDA and ion accumulation and a higher level of proline, relative water content, root/shoot ration, enzymatic activities of ROS scavengers and upregulation of stress-responsive genes. Based on the relative expression of stress-responsive genes and ionic content, the working model highlights the role of PsLecRLK in the extrusion of Na+ ion from the cell. This extrusion of Na+ ion is facilitated by higher expression of SOS1 (Na+/K+ channel) in transgenic plants as compared to WT plants. Altered expression of stress-responsive genes and change in biochemical and physiological properties of the cell suggests an extensive reprogramming of the stress-responsive metabolic pathways by PsLecRLK under stress condition, which could be responsible for the salt tolerance capability.

Stress-induced Oryza sativa RuvBL1a is DNA-independent ATPase and unwinds DNA duplex in 3' to 5' direction.

RuvB, a member of AAA+ (ATPases Associated with diverse cellular Activities) superfamily of proteins, is essential, highly conserved and multifunctional in nature as it is involved in DNA damage repair, mitotic assembly, switching of histone variants and assembly of telomerase core complex. RuvB family is widely studied in various systems such as Escherichia coli, yeast, human, Drosophila, Plasmodium falciparum and mouse, but not well studied in plants. We have studied the transcript level of rice homologue of RuvB gene (OsRuvBL1a) under various abiotic stress conditions, and the results suggest that it is upregulated under salinity, cold and heat stress. Therefore, the OsRuvBL1a protein was characterized using in silico and biochemical approaches. In silico study confirmed the presence of all the four characteristic motifs of AAA+ superfamily-Walker A, Walker B, Sensor I and Sensor II. Structurally, OsRuvBL1a is similar to RuvB1 from Chaetomium thermophilum. The purified recombinant OsRuvBL1a protein shows unique DNA-independent ATPase activity. Using site-directed mutagenesis, the importance of two conserved motifs (Walker B and Sensor I) in ATPase activity has been also reported with mutants D302N and N332H. The OsRuvBL1a protein unwinds the duplex DNA in the 3' to 5' direction. The presence of unique DNA-independent ATPase and DNA unwinding activities of OsRuvBL1a protein and upregulation of its transcript under abiotic stress conditions suggest its involvement in multiple cellular pathways. The first detailed characterization of plant RuvBL1a in this study may provide important contribution in exploiting the role of RuvB for developing the stress tolerant plants of agricultural importance.

Prediction and validation of cis-regulatory elements in 5' upstream regulatory regions of lectin receptor-like kinase gene family in rice.

Lectin receptor-like kinases (LecRLKs) play crucial roles in regulating plant growth and developmental processes in response to stress. In transcriptional gene regulation for normal cellular functions, cis-acting regulatory elements (CREs) direct the temporal and spatial gene expression with respect to environmental stimuli. A complete insightful of the transcriptional gene regulation system relies on effective functional analysis of CREs. Here, we analyzed the potential putative CREs present in the promoters of rice LecRLKs genes by using PlantCARE database. The CREs in LecRLKs promoters are associated with plant growth/development, light response, plant hormonal regulation processes, various stress responses, hormonal response like ABA, root-specific expression responsive, drought responsive, and cell and organ specific regulatory elements. The effect of methylation on these cis-regulatory elements was also analyzed. Real-time analysis of rice seedling under various stress conditions showed the expression levels of selected LecRLK genes superimposing the number of different CREs present in 5' upstream region. The overall results showed that the possible CREs function in the selective expression/regulation of LecRLKs gene family and during rice plant development under stress.

Assessing zygosity in progeny of transgenic plants: current methods and perspectives.

Homozygosity is highly desirable in transgenic plants research to ensure the stable integration and inheritance of transgene(s). Simple, reliable and high-throughput techniques to detect the zygosity of transgenic events in plants are invaluable tools for biotechnology and plant breeding companies. Currently, a number of basic techniques are being used to determine the zygosity of transgenic plants in T1 generation. For successful application of any technique, precision and simplicity of approach combined with the power of resolution are important parameters. On the basis of simplicity, resolution and cost involved, the available techniques have been classified into three major classes which are conventional methods, current methods and next generation methods. Conventional methods include antibiotic marker-based selection and the highly labor intensive Southern blot analysis. In contrast, methods such as real time PCR, TAIL PCR and competitive PCR are not only cost effective but rapid as well. Moreover, methods such as NGS, digital PCR and loop-mediated isothermal amplification also provide a cost effective, fast and not so labor intensive substitute of current methods. In this review, we have attempted to compare and contrast all the available efficient methods to distinguish homozygous plants in progeny of transgenics. This review also provides information of various techniques available for determining zygosity in plants so as to permit researchers to make informed choices of techniques that best suit their analyses. More importantly, detection and subsequent selection of homozygous individuals is central for facilitating the movement of transgenic plants from the laboratory to the field.