Biomedical subjects
Publications and source records attributed to Jian-Kang Zhu.
Systematic comparison of Cas12i gene-editing tools in tomato identifies Cas-SF01 as the most efficient variant, achieving editing efficiencies comparable to conventional Cas9 at some genomic sites and providing a compact, highly specific, and versatile tool for studying gene function, engineering fruit metabolism, and developing improved tomato varieties through precision breeding.
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This study introduces an intronic artificial microRNA (IamiRNA) strategy that combines CRISPR-Cas9-mediated knock-in with endogenous miRNA processing for targeted gene silencing in plants. By inserting amiRNA precursors into introns of endogenous genes, this approach enables effective, tissue-specific gene silencing without persistent transgene expression, offering a promising tool for functional genomics and crop improvement.
TnpB, a compact RNA-guided nuclease ancestral to Cas12, is optimized for plant genome editing. A 99-nt enhanced RNA and T5 exonuclease fusion generate TnpBe5, boosting rice editing 2.5-fold. Coupling with a single-strands annealing (SSA)-responsive HYYG surrogate (TnpBmax) enriches edited cells, achieving up to 81.5% efficiency and high homozygosity across rice and tomato.
A stable and efficient transformation system is crucial for functional genomics and trait improvement in soybean. This study developed a tissue culture based genetic transformation system incorporating dual selection (Spectinomycin and RUBY). This system significantly enhances transformation efficiency, shortens the transformation cycle, and demonstrates broad genotype independence, providing a powerful tool for soybean research and breeding.
Spontaneous gain or loss of DNA methylation occurs in plant and animal genomes, and DNA methylation changes can lead to meiotically stable epialleles that generate heritable phenotypic diversity. However, it is unclear whether transgenerational epigenetic stability may be regulated by any cellular factors. Here, we examined spontaneously occurring variations in DNA methylation in wild-type and ros1 mutant Arabidopsis plants that were propagated for ten generations from single-seed descent. We found that the ros1 mutant, which is defective in active DNA demethylation, showed an increased transgenerational epimutation rate. The ros1 mutation led to more spontaneously gained methylation than lost methylation at individual cytosines, compared to the wild type which had similar numbers of spontaneously gained and lost methylation cytosines. Consistently, transgenerational differentially methylated regions were also biased toward hypermethylation in the ros1 mutant. Our results reveal a genetic contribution of the ROS1 DNA demethylase to transgenerational epigenetic stability and suggest that ROS1 may have an unexpected surveillance function in preventing transgenerational DNA methylation increases.
The Arabidopsis DNA glycosylase/lyase ROS1 participates in active DNA demethylation by a base-excision pathway. ROS1 has been shown to be required for demethylating a transgene promoter. To determine the function of ROS1 in demethylating endogenous loci, we carried out bisulfite-sequencing analysis of several transposons and other genes in the ros1 mutant. In the wild-type, although CpG sites at the majority of these loci are heavily methylated, many of the CpXpG and CpXpX sites have low levels of methylation or are not at all methylated. However, these CpXpG and CpXpX sites become heavily methylated in the ros1 mutant. Associated with this increased DNA methylation, these loci show decreased expression in the ros1 mutant. Our results suggest that active DNA demethylation is important in pruning the methylation patterns of the genome, and even the normally "silent" transposons are under dynamic control by both methylation and demethylation. This dynamic control may be important in keeping the plant epigenome plastic so that it can efficiently respond to developmental and environmental cues.
Protein phosphatase 2Cs (PP2Cs) constitute a major class of phosphatases in plants. PP2Cs play important roles in many signaling pathways by countering the action of specific protein kinases. In addition to their role in several environmental stress-related signal transduction pathways, they are also involved in plant metabolism. Protein phosphatases often physically associate with their protein kinase counterparts. One approach to understanding PP2C function is to identify their interacting protein kinases. We describe a yeast two-hybrid assay system used in our lab to determine the interaction between members of the PP2C family and protein kinases in the SOS2 family. This chapter and the cited articles describing related work might be of help in discovering interactions between other protein phosphatases and kinases.
The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.
TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.
Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 A (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants.
C(2)H(2)-zinc finger proteins that contain the EAR repressor domain are thought to play a key role in modulating the defense response of plants to abiotic stress. Constitutive expression of the C(2)H(2)-EAR zinc finger protein Zat10 in Arabidopsis was found to elevate the expression of reactive oxygen-defense transcripts and to enhance the tolerance of plants to salinity, heat and osmotic stress. Surprisingly, knockout and RNAi mutants of Zat10 were also more tolerant to osmotic and salinity stress. Our results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.
RNA interference, mediated by small interfering RNAs (siRNAs), is a conserved regulatory process that has evolved as an antiviral defense mechanism in plants and animals. It is not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. Here, we report the discovery of an endogenous siRNA, nat-siRNAATGB2, that is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2. We demonstrate that the biogenesis of this siRNA requires DCL1, HYL1, HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the cognate host disease resistance gene RPS2 and the NDR1 gene that is required for RPS2-specified resistance. This siRNA contributes to RPS2-mediated race-specific disease resistance by repressing PPRL, a putative negative regulator of the RPS2 resistance pathway.
To study the genetic control of plant responses to cold stress, Arabidopsis thaliana mutants were isolated by a screen for mutations that impair cold-induced transcription of the CBF3-LUC reporter gene. We report here the characterization and cloning of a mutated gene, atnup160-1, which causes reduced CBF3-LUC induction under cold stress. atnup160-1 mutant plants display altered cold-responsive gene expression and are sensitive to chilling stress and defective in acquired freezing tolerance. AtNUP160 was isolated through positional cloning and shown to encode a putative homolog of the animal nucleoporin Nup160. In addition to the impaired expression of CBF genes, microarray analysis revealed that a number of other genes important for plant cold tolerance were also affected in the mutants. The atnup160 mutants flower early and show retarded seedling growth, especially at low temperatures. AtNUP160 protein is localized at the nuclear rim, and poly(A)-mRNA in situ hybridization shows that mRNA export is defective in the atnup160-1 mutant plants. Our study suggests that Arabidopsis AtNUP160 is critical for the nucleocytoplasmic transport of mRNAs and that it plays important roles in plant growth and flowering time regulation and is required for cold stress tolerance.
The adverse effects of high salt on plants include Na(+) toxicity and hyperosmotic and oxidative stresses. The plasma membrane-localized Na(+)/H(+) antiporter SOS1 functions in the extrusion of toxic Na(+) from cells and is essential for plant salt tolerance. We report here that, under salt or oxidative stress, SOS1 interacts through its predicted cytoplasmic tail with RCD1, a regulator of oxidative-stress responses. Without stress treatment, RCD1 is localized in the nucleus. Under high salt or oxidative stress, RCD1 is found not only in the nucleus but also in the cytoplasm. Like rcd1 mutants, sos1 mutant plants show an altered sensitivity to oxidative stresses. The rcd1mutation causes a decrease in salt tolerance and enhances the salt-stress sensitivity of sos1 mutant plants. Several genes related to oxidative-stress tolerance were found to be regulated by both RCD1 and SOS1. These results reveal a previously uncharacterized function of a plasma membrane Na(+)/H(+) antiporter in oxidative-stress tolerance and shed light on the cross-talk between the ion-homeostasis and oxidative-stress detoxification pathways involved in plant salt tolerance.
Cold temperatures trigger the expression of the CBF family of transcription factors, which in turn activate many downstream genes that confer freezing tolerance to plants. It has been shown previously that the cold regulation of CBF3 involves an upstream bHLH-type transcription factor, ICE1. ICE1 binds to the Myc recognition sequences in the CBF3 promoter. Apart from Myc recognition sequences, CBF promoters also have Myb recognition sequences. We report here that the Arabidopsis MYB15 is involved in cold-regulation of CBF genes and in the development of freezing tolerance. The MYB15 gene transcript is up-regulated by cold stress. The MYB15 protein interacts with ICE1 and binds to Myb recognition sequences in the promoters of CBF genes. Overexpression of MYB15 results in reduced expression of CBF genes whereas its loss-of-function leads to increased expression of CBF genes in the cold. The myb15 mutant plants show increased tolerance to freezing stress whereas its overexpression reduces freezing tolerance. Our results suggest that MYB15 is part of a complex network of transcription factors controlling the expression of CBFs and other genes in response to cold stress.
A number of protein and RNA-processing mutants have been shown to affect ABA sensitivity. A new mutant, sad2-1, was isolated from a T-DNA mutagenized population of RD29A:LUC plants and shown to have increased luminescence after ABA, salt, cold or polyethylene glycol treatments. Expression of several ABA- and stress-responsive genes was higher in the mutant than in the wild type. sad2-1 also exhibited ABA hypersensitivity in seed germination and seedling growth. SAD2 was found to encode an importin beta-domain family protein likely to be involved in nuclear transport. SAD2 was expressed at a low level in all tissues examined except flowers, but SAD2 expression was not inducible by ABA or stress. Subcellular localization of GFP-tagged SAD2 showed a predominantly nuclear localization, consistent with a role for SAD2 in nuclear transport. Knockout of the closest importin beta homolog of SAD2 in Arabidopsis did not duplicate the sad2 phenotype, indicating that SAD2 plays a specific role in ABA signaling. Analysis of RD29A:LUC luminescence and ABA and stress sensitivity in double mutants of sad2-1 and sad1 or abh1-7, a newly isolated allele of ABH1 also in the RD29A:LUC background, suggested that SAD2 acts upstream of or has additive effects with these two genes. The results suggest a role for nuclear transport in ABA signal transduction, and the possible roles of SAD2 in relation to that of SAD1 and ABH1 are discussed.
DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive beta- and delta-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis.