Cross-resistance, inheritance and biochemical mechanism of abamectin resistance in a field-derived strain of the citrus red mite, Panonychus citri (Acari: Tetranychidae).

BACKGROUND: The citrus red mite, Panonychus citri (McGregor), a global pest of citrus, has developed different levels of resistance to various acaricides in the field. Abamectin is one of the most important insecticides/acaricides worldwide, targetting a wide number of insect and mite pests. The evolution of abamectin resistance in P. citri is threatening the sustainable use of abamectin for mite control. RESULTS: The abamectin resistant strain (NN-Aba), derived from a field strain NN by consistent selection with abamectin, showed 4279-fold resistance to abamectin compared to a relatively susceptible strain (SS) of P. citri. Cross-resistance of NN-Aba was observed between abamectin and emamectin benzoate, pyridaben, fenpropathrin and cyflumetofen. Inheritance analyses indicated that abamectin resistance in the NN-Aba strain was autosomal, incompletely recessive and polygenic. The synergy experiment showed that abamectin toxicity was synergized by piperonyl butoxide (PBO), diethyl maleate (DEM) and tributyl phosphorotrithiotate (TPP) in the NN-Aba strain, and synergy ratios were 2.72-, 2.48- and 2.13-fold, respectively. The glutathione-S-transferases activity in the NN-Aba strain were significantly increased by 2.08-fold compared with the SS strain. CONCLUSION: The abamectin resistance was autosomal, incompletely recessive and polygenic in P. citri. The NN-Aba strain showed cross-resistance to various acaricides with different modes of action. Metabolic detoxification mechanism participated in abamectin resistance in NN-Aba strain. These findings provide useful information for resistance management of P. citri in the field. © 2023 Society of Chemical Industry.

Correction: Liu et al. LeGRXS14 Reduces Salt Stress Tolerance in Arabidopsis thaliana. Plants 2023, 12, 2320.

The authors wish to correct the following error in the original paper [...].

CYP4CL2 Confers Metabolic Resistance to Pyridaben in the Citrus Pest Mite Panonychus citri.

The citrus red mite Panonychus citri has developed strong resistance to acaricides. Cytochrome P450 monooxygenases (P450s) can detoxify pesticides and are involved in pesticide resistance in many insects. Here, a pyridaben-resistant P. citri strain showed cross-resistance to cyenopyrafen, bifenazate, fenpyroximate, and tolfenpyrad. Piperonyl butoxide, a P450 inhibitor, significantly increased the toxicity of pyridaben to resistant (Pyr_Rs) and susceptible (Pyr_Control) P. citri strains. P450 activity was significantly higher in Pyr_Rs than in Pyr_Control. Analyses of RNA-Seq data identified a P450 gene (CYP4CL2) that is potentially involved in pyridaben resistance. Consistently, it was up-regulated in two field-derived resistant populations (CQ_WZ and CQ_TN). RNA interference-mediated knockdown of CYP4CL2 significantly decreased the pyridaben resistance in P. citri. Transgenic Drosophila melanogaster expressing CYP4CL2 showed increased pyridaben resistance. Molecular docking analysis showed that pyridaben could bind to several amino acids at substrate recognition sites in CYP4CL2. These findings shed light on P450-mediated pyridaben resistance in pest mites.

LeGRXS14 Reduces Salt Stress Tolerance in Arabidopsis thaliana.

Salt stress represents a significant abiotic stressor for plants and poses a severe threat to agricultural productivity. Glutaredoxins (GRXs) are small disulfide reductases that can scavenge cellular reactive oxygen species and are crucial for plant growth and development, particularly under stressful circumstances. Although CGFS-type GRXs were found to be involved in various abiotic stresses, the intrinsic mechanism mediated by LeGRXS14, a tomato (Lycopersicon esculentum Mill.) CGFS-type GRX, is not yet fully understood. We discovered that LeGRXS14 is relatively conserved at the N-terminus and exhibits an increase in expression level under salt and osmotic stress conditions in tomatoes. The expression levels of LeGRXS14 in response to osmotic stress peaked relatively rapidly at 30 min, while the response to salt stress only peaked at 6 h. We constructed LeGRXS14 overexpression Arabidopsis thaliana (OE) lines and confirmed that LeGRXS14 is located on the plasma membrane, nucleus, and chloroplasts. In comparison to the wild-type Col-0 (WT), the OE lines displayed greater sensitivity to salt stress, resulting in a profound inhibition of root growth under the same conditions. Analysis of the mRNA levels of the WT and OE lines revealed that salt stress-related factors, such as ZAT12, SOS3, and NHX6, were downregulated. Based on our research, it can be concluded that LeGRXS14 plays a significant role in plant tolerance to salt. However, our findings also suggest that LeGRXS14 may act as a negative regulator in this process by exacerbating Na+ toxicity and the resulting oxidative stress.

Resistance of Panonychus citri (McGregor) (Acari: Tetranychidae) to pyridaben in China: monitoring and fitness costs.

BACKGROUND: Panonychus citri is a major citrus pest worldwide. The short life cycle and high reproductive potential of P. citri, combined with heavy acaricide use, have led to high levels of resistance to acaricides, posing a threat to global resistance management programs. Here, resistance monitoring was established to determine the pyridaben resistance status of ten P. citri populations in China from 2014 to 2021 using a leaf-dipping assay. Four characterized strains-the susceptible strain (Lab_S), the resistant strain (Pyr_R), as well as the segregated resistant strain (Pyr_Rs) and the segregated susceptible strain (Pyr_Control) derived from the crossing of the Lab_S and Pyr_R strains, were used to evaluate the life-history characteristics using age-stage, two-sex life tables. RESULTS: Most P. citri populations developed high resistance to pyridaben. Resistance levels exceeded 1000-fold in Yuxi, Anyue, Nanning, and Ganzhou populations compared with the Lab_S strain. Compared with Pyr_Control, two key fitness cost criteria, developmental period and fecundity, showed significant differences in Pyr_Rs under consistent conditions. The intrinsic rate of increase, net reproductive rate and gross reproductive rate were lower in the resistant strain compared with the Pyr_Control strain. The Pyr_Rs strain had a lower relative fitness of 0.934 compared with the Pyr_Control. Moreover, the life-history traits and population parameters of the Pyr_R strain also showed significant differences compared with the Lab_S strain. CONCLUSION: The resistance levels to pyridaben varied greatly among the different P. citri populations and showed regional differences. Substantial fitness costs are associated with pyridaben resistance. This study provides potential implications for developing strategies for resistance management in P. citri. © 2022 Society of Chemical Industry.

The Role of Chloride Channels in Plant Responses to NaCl.

Chloride (Cl-) is considered a crucial nutrient for plant growth, but it can be a challenge under saline conditions. Excessive accumulation of Cl- in leaves can cause toxicity. Chloride channels (CLCs) are expressed in the inner membranes of plant cells and function as essential Cl- exchangers or channels. In response to salt stress in plants, CLCs play a crucial role, and CLC proteins assist in maintaining the intracellular Cl- homeostasis by sequestering Cl- into vacuoles. Sodium chloride (NaCl) is the primary substance responsible for causing salt-induced phytotoxicity. However, research on plant responses to Cl- stress is comparatively rare, in contrast to that emphasizing Na+. This review provides a comprehensive overview of the plant response and tolerance to Cl- stress, specifically focusing on comparative analysis of CLC protein structures in different species. Additionally, to further gain insights into the underlying mechanisms, the study summarizes the identified CLC genes that respond to salt stress. This review provides a comprehensive overview of the response of CLCs in terrestrial plants to salt stress and their biological functions, aiming to gain further insights into the mechanisms underlying the response of CLCs in plants to salt stress.

A microRNA-microRNA crosstalk network inferred from genome-wide single nucleotide polymorphism variants in natural populations of Arabidopsis thaliana.

To adapt to variable natural conditions, plants have evolved several strategies to respond to different environmental stresses. MicroRNA (miRNA)-mediated gene regulation is one of such strategies. Variants, e.g., single nucleotide polymorphisms (SNPs) within the mature miRNAs or their target sites may cause the alteration of regulatory networks and serious phenotype changes. In this study, we proposed a novel approach to construct a miRNA-miRNA crosstalk network in Arabidopsis thaliana based on the notion that two cooperative miRNAs toward common targets are under a strong pressure to be inherited together across ecotypes. By performing a genome-wide scan of the SNPs within the mature miRNAs and their target sites, we defined a "regulation fate profile" to describe a miRNA-target regulation being static (kept) or dynamic (gained or lost) across 1,135 ecotypes compared with the reference genome of Col-0. The cooperative miRNA pairs were identified by estimating the similarity of their regulation fate profiles toward the common targets. The reliability of the cooperative miRNA pairs was supported by solid expressional correlation, high PPImiRFS scores, and similar stress responses. Different combinations of static and dynamic miRNA-target regulations account for the cooperative miRNA pairs acting on various biological characteristics of miRNA conservation, expression, homology, and stress response. Interestingly, the targets that are co-regulated dynamically by both cooperative miRNAs are more likely to be responsive to stress. Hence, stress-related genes probably bear selective pressures in a certain group of ecotypes, in which miRNA regulations on the stress genes reprogram. Finally, three case studies showed that reprogramming miRNA-miRNA crosstalk toward the targets in specific ecotypes was associated with these ecotypes' climatic variables and geographical locations. Our study highlights the potential of miRNA-miRNA crosstalk as a genetic basis underlying environmental adaptation in natural populations.

Molting process revealed by the detailed expression profiles of RXR1/RXR2 and mining the associated genes in a spider mite, Panonychus citri.

Spider mites have one ecdysone receptor (EcR) and multiple retinoid X receptors (RXRs). However, the function of these RXRs in spider mite development is unknown. Here, we screened the expression dynamics of two PcRXR isoforms at 4 h intervals in the deutonymphal stage of Panonychus citri. The results showed that PcEcR had an expression pattern similar to that of PcRXR2. For PcRXR1, its expression remained at a certain high level, when there was a decrease of both PcEcR and PcRXR2. In situ hybridization showed that PcRXR2 was detected in the central nervous mass, while the ecdysteroid biosynthesis gene PcSpo was mainly expressed at the edge of the central nervous mass. RNAi-based silencing of PcRXR1 or PcRXR2 showed the same phenotype as in mites with that of silencing PcEcR. Furthermore, RNA-seq was used to mine the genes associated with the expression dynamics of PcRXR1 or PcRXR2, which revealed that the heterodimer of EcR-RXR2 in spider mites might be linked with the cell autophagy and tissue remodeling during apolysis, and RXR1 might be linked with new epicuticle and exocuticle secretion during ecdysis. Taken together, these results increase our understanding of the regulation mechanism of ecdysteroid signal pathway in spider mite development.

De novo RNA-Seq and Annotation of Sesquiterpenoid and Ecdysteroid Biosynthesis Genes and MicroRNAs in a Spider Mite Eotetranychus kankitus.

Eotetranychus kankitus is an important mite pest in citrus, but molecular data on the developmental processes of E. kankitus are lacking. The different development stages mix of E. kankitus was used to sequence for transcriptome and small RNAs to identify genes and predict miRNAs associated with sesquiterpenoid and ecdysteroid biosynthesis and signaling pathways. More than 36 million clean reads were assembled and 67,927 unigenes were generated. Of the unigenes, 19,300 were successfully annotated through annotation databases NR, SwissProt, COG, GO, KEGG, PFAM, and KOG. The transcripts were involved in sesquiterpenoid biosynthesis (11 genes) and ecdysteroid biosynthesis and signaling pathway (13 genes). Another, small RNA library was obtained and 31 conserved miRNAs were identified. Five most abundant miRNAs were Ek-miR-5735, Ek-miR-1, Ek-miR-263a, Ek-miR-184, and Ek-miR-8. The target genes related to sesquiterpenoid and ecdysteroid showed that 10 of the conserved miRNAs could potentially target the sesquiterpenoid and ecdysteroid pathway according to four-prediction software, sRNAT, miRanda, RNAhybrid, and Risearch2. Thus, the results of this study will provide bioinformatics information for further molecular studies of E. kankitus which may facilitate improved pest control strategies.

Genome-Wide Characterization and Identification of Long Non-Coding RNAs during the Molting Process of a Spider Mite, Panonychus citri.

Molting is essential for arthropods to grow. As one of the important arthropod pests in agriculture, key spider mite species (Tetranychus and Panonychus) can normally molt three times from the larva to adult stage within a week. This physiological strategy results in the short lifecycle of spider mites and difficulties in their control in the field. Long non-coding RNAs (lncRNAs) regulate transcriptional editing, cellular function, and biological processes. Thus, analysis of the lncRNAs in the spider mite molting process may provide new insights into their roles in the molting mechanism. For this purpose, we used high-throughput RNA-seq to examine the expression dynamics of lncRNAs and mRNAs in the molting process of different development stages in Panonychus citri. We identified 9199 lncRNAs from 18 transcriptomes. Analysis of the lncRNAs suggested that they were shorter and had fewer exons and transcripts than mRNAs. Among these, 356 lncRNAs were differentially expressed during three molting processes: late larva to early protonymph, late protonymph to early deutonymph, and late deutonymph to early adult. A time series profile analysis of differentially expressed lncRNAs showed that 77 lncRNAs were clustered into two dynamic expression profiles (Pattern a and Pattern c), implying that lncRNAs were involved in the molting process of spider mites. Furthermore, the lncRNA-mRNA co-expression networks showed that several differentially expressed hub lncRNAs were predicted to be functionally associated with typical molting-related proteins, such as cuticle protein and chitin biosynthesis. These data reveal the potential regulatory function of lncRNAs in the molting process and provide datasets for further analysis of lncRNAs and mRNAs in spider mites.

RNAi of the nuclear receptor HR3 suggests a role in the molting process of the spider mite Panonychus citri.

Ecdysteroids regulate molting in arthropods by binding to heterodimers of the ecdysone receptor and retinoid-X-receptor, homologous to the ultraspiracle protein, to induce the expression of downstream signal response genes including the nuclear receptor HR3. However, the detailed expression dynamics of HR3 during molting in spider mites are not yet clear. In this study, the full length of PcHR3 was retrieved based on the genome of citrus red mite, Panonychus citri. The open reading frame is 1707 bp encoding 568 amino acids, which contains a DNA binding domain and a ligand binding domain. Then, the expression pattern of PcHR3 was analyzed throughout the development of the deutonymph by RT-qPCR. The result showed that PcHR3 was mainly transcribed in the late deutonymph stage, when the deutonymph was at least 24 h old and motionless, the critical point at which the mites started molting. Transcription reached the highest level in 32-h-old deutonymphs and decreased by 36 h, where the mites remained in a quiescent state. Further silencing of PcHR3 by leaf-disc-based delivery of dsRNA to 8-h-old deutonymph mites, resulted in retarded development and death of 58% of deutonymphs. In summary, we suggest that PcHR3 regulates the latter stages of molting in P. citri.

Expression dynamics of key ecdysteroid and juvenile hormone biosynthesis genes imply a coordinated regulation pattern in the molting process of a spider mite, Tetranychus urticae.

In insects, the ecdysteroid 20-hydroxyecdysone coordinates with juvenile hormone (JH) to regulate the process of molting, development and metamorphosis; however, this interaction is still unclear in the mites. In this study, we investigated the gene related to ecdysteroid and JH biosynthesis pathways, including four ecdysteroid and 11 JH biosynthesis genes. We examined their expression patterns during molting of different developmental stages of the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), an important agricultural pest that feeds on more than 1100 plant species. The expression of ecdysteroid biosynthesis Halloween genes exhibited a positive zigzag-like pattern, with a peak after 8 h of molting and a drop 8 h after entering each quiescent stage. In contrast, JH biosynthesis genes expression displayed a negative zigzag-like pattern, with a peak at 8 h after entering each quiescent stage and a drop after 8 h of each molting. These opposite patterns imply that ecdysteroid and JH expression is coordinated during the developmental transition. Our data provide an initial perspective on the co-expression of ecdysteroid and JH biosynthesis genes to regulate this important developmental process in the two-spotted spider mite.

Heterologous Expression and Functional Analysis of Rice GLUTAMATE RECEPTOR-LIKE Family Indicates its Role in Glutamate Triggered Calcium Flux in Rice Roots.

BACKGROUND: Tremendous progress has been made in understanding the functions of the GLUTAMATE RECEPTOR-LIKE (GLR) family in Arabidopsis. Still, the functions of OsGLRs in rice, especially the ion channel activities, are largely unknown. RESULTS: Using the aequorin-based luminescence imaging system, we screened the specificity of amino acids involved in the induction of Ca(2+) flux in rice roots. Of all the amino acids tested, glutamate (Glu) was the only one to trigger Ca(2+) flux significantly in rice roots. Detailed analysis showed a dose response of Ca(2+) increase to different concentrations of Glu. In addition, the Ca(2+) spike response to Glu was rapid, within 20 s after the application. A desensitization assay and pharmacological tests showed that the Glu-triggered Ca(2+) flux is mediated by OsGLRs. Whole genome analysis identified 13 OsGLR genes in rice, and these genes have various expression patterns in different tissues. Subcellular localization studies showed that all the OsGLRs examined are likely localized to the plasma membrane. Bacteria growth assays showed that at least OsGLR2.1 and OsGLR3.2 have the potential to mediate ion uptake in bacteria. Further analysis using Fura-2-based Ca(2+) imaging revealed a Glu-triggered Ca(2+) increase in OsGLR2.1-expressing human embryonic kidney (HEK) cells. CONCLUSIONS: Our work provides a molecular basis for investigating mechanisms of Glu-triggered Ca(2+) flux in rice.

Identification of miRNAs associated with dark-induced senescence in Arabidopsis.

BACKGROUND: microRNAs (miRNAs) are endogenous small (~21 nucleotide) single-stranded non-coding RNAs that typically function by guiding cleavage of target genes. To find the miRNAs that may be involved in dark-induced leaf senescence, we identified miRNAs by microarray platform using Arabidopsis thaliana leaves from both whole darkened plants (DPs) and individually darkened leaves (IDLs). RESULTS: We found that the expressions of 137 miRNAs (P < 0.01, signal intensity >0) were significantly changed both in DP and IDL leaves. Among them, the expression levels of 44 miRNAs were relative higher than others (P < 0.01, signal intensity > 500). Of these differentially expressed miRNAs, 6 miRNAs (miR319a, 319c, miR159, miR164a, miR164c and miR390a) have been previously reported to be involved in dark-induced leaf senescence, and the remaining 38 miRNAs have not been implicated in leaf senescence before. Target genes of all 44 miRNAs were predicted, and some of them, such as NAC1, At3g28690, At2g17640 and At2g45160, were found in the Leaf Senescence Database (LSD). GO and KEGG analysis of 137 miRNAs showed that the predicted target genes were significantly enriched in transcription regulation, development-related biological processes and metabolic pathways. Expression levels of some of the corresponding miRNA targets (At1g73440, At2g03220 and At5g54810) were analysed and found to be significantly different in DP/IDL than that in WT. CONCLUSIONS: A microarray analysis about dark-induced miRNAs involved in leaf senescence are present here. Further expression analysis revealed that some new founding miRNAs maybe regulate leaf senescence in Arabidopsis, and the findings highlight the important role of miRNAs in dark-induced leaf senescence.

Identification of miRNAs and their targets using high-throughput sequencing and degradome analysis in cytoplasmic male-sterile and its maintainer fertile lines of Brassica juncea.

BACKGROUND: Regulatory network of cytoplasmic male sterility (CMS) occurrence is still largely unknown in plants, although numerous researches have been attempted to isolate genes involved in CMS. Here, we employed high-throughput sequencing and degradome analysis to identify microRNAs and their targets using high-throughput sequencing in CMS and its maintainer fertile (MF) lines of Brassica juncea. RESULTS: We identified 197 known and 78 new candidate microRNAs during reproductive development of B. juncea. A total of 47 differentially expressed microRNAs between CMS and its MF lines were discovered, according to their sequencing reads number. Different expression levels of selected microRNAs were confirmed by using real-time quantitative PCR between CMS and MF lines. Furthermore, we observed that the transcriptional patterns of these microRNAs could be mimicked by artificially inhibiting mitochondrial F1F0-ATPase activity and its function in MF line by using treatment with oligomycin. Targeted genes of the microRNAs were identified by high-throughput sequencing and degradome approaches, including auxin response factor, NAC (No Apical Meristem) domain transcription factor, GRAS family transcription factor, MYB transcription factor, squamosa promoter binding protein, AP2-type transcription factor, homeobox/homeobox-leucine zipper family and TCP family transcription factors, which were observed to be differentially expressed between CMS and MF. CONCLUSION: Taken together, from these findings we suggested microRNA might participate in the regulatory network of CMS by tuning fork in gene expressions in CMS B. juncea. The differential expression of miRNAs observed between CMS and MF lines suggested that biogenesis of miRNAs could be influenced in the CMS.

Reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in cytoplasmic male-sterile Brassica juncea.

The signal from organelle to nucleus, namely retrograde regulation of nuclear gene expression, was largely unknown. Due to the nuclear-cytoplasmic incompatibility in cytoplasmic male-sterile (CMS) plants, we employed CMS Brassica juncea to investigate the retrograde regulation of nuclear gene expression in this study. We studied how reduced BjRCE1 gene expression caused by the nuclear-cytoplasmic incompatibility altered the auxin response in CMS of B. juncea. We isolated the BjRCE1 gene that was located in the nucleus from B. juncea. Over-expression of BjRCE1 enhanced auxin response in transgenic Arabidopsis. The expression of BjRCE1 was significantly reduced in CMS compared with its maintainer fertile (MF) line of B. juncea. There were fewer lateral roots in CMS than MF under normal and treatment of indole-3-acetic acid (IAA) conditions. Expression patterns of several auxin-related genes together with their phenotypes indicated a reduced auxin response in CMS compared to MF. The phenotypes of auxin response and auxin-related gene expression pattern could be mimicked by inhibiting mitochondrial function in MF. Taken together, we proposed reduced expression of BjRCE1 gene modulated by nuclear-cytoplasmic incompatibility alters auxin response in CMS B. juncea. This may be an important mechanism of retrograde regulation of nuclear gene expression in plants.

Reduced expression of CTR1 gene modulated by mitochondria causes enhanced ethylene response in cytoplasmic male-sterile Brassica juncea.

We studied how mitochondria affect ethylene response via modulation of CTR1 expression in cytoplasmic male-sterile (CMS) Brassica juncea. The expression of CTR1 gene was reduced in CMS compared with male-fertile (MF) lines. We observed that hypocotyl and root lengths were shorter than in the MF line during germination in the dark. An enhanced ethylene response was observed in CMS plants as shown by the CMS and maintainer line phenotypes treated with 1-aminocyclopropane-1-carboxylic acid. The phenotype in CMS plants could be recovered to the maintainer line when treated with Ag(+) . One ethylene response gene, plant defensin gene, was detected to be induced in CMS. The behavior of this phenotype could be mimicked by treating the maintainer line with antimycin A that disturbs mitochondrial function, which showed reduced length of hypocotyl and roots, and resulted in similar expression patterns of ethylene-related genes as in CMS. The reduced length of hypocotyl and roots could be recovered to the maintainer line by treatment with gibberellic acid (GA(3) ). In addition, the GA(3) content was reduced in CMS plants and in the MF line treated with antimycin A. Ethylene treatment markedly affects GA(3) content; however, GA(3) did not significantly affect ethylene-related gene expression in regards to regulation of hypocotyl and root length, which suggests that ethylene acts upstream via gibberellin to regulate hypocotyls and root development. Taken together, our results suggest a link between mitochondrial modulation of the ethylene and gibberellin pathway that regulates the development of hypocotyl and roots.

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea.

BACKGROUND: The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility. RESULTS: It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the ß subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the ß subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered. CONCLUSION: The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through retrograde regulation of nuclear gene expression in Brassica juncea.

The L279P mutation of nuclear distribution gene C (NudC) influences its chaperone activity and lissencephaly protein 1 (LIS1) stability.

LIS1, a gene mutated in classical lissencephaly, plays essential roles in cytoplasmic dynein regulation, mitosis and cell migration. However, the regulation of LIS1 (lissencephaly protein 1) protein remains largely unknown. Genetic studies in Aspergillus nidulans have uncovered that the Nud (nuclear distribution) pathway is involved in the regulation of cytoplasmic dynein complex and a temperature-sensitive mutation in the nudC gene (L146P) greatly reduces the protein levels of NudF, an Aspergillus ortholog of LIS1. Here, we showed that L146 in Aspergillus NudC and its flanking region were highly conservative during evolution. The similar mutation in human NudC (L279P) obviously led to reduced LIS1 and cellular phenotypes similar to those of LIS1 down-regulation. To explore the underlying mechanism, we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90, which is also associated with LIS1. Inhibition of Hsp90 chaperone function by either geldanamycin or radicicol resulted in a decrease in LIS1 levels. Ectopic expression of Hsp90 partially reversed the degradation of LIS1 caused by overexpression of NudC-L279P. Furthermore, NudC was found to regulate the ATPase activity of Hsp90, which was repressed by the mutation of L279P. Interestingly, NudC itself was shown to possess a chaperone function, which also was suppressed by the L279P mutation. Together, these data suggest that NudC may be involved in the regulation of LIS1 stability by its chaperone function.

Percutaneous coagulation therapy of hepatocellular carcinoma by combining microwave coagulation therapy and ethanol injection.

OBJECTIVE: To determine if larger hepatocellular carcinoma (HCC) coagulation volumes can be obtained by combining percutaneous microwave coagulation therapy (PMCT) and ethanol injection (PEI) or PEI followed by PMCT with occlusion of the feeding artery. SUBJECTS AND METHODS: Eighty patients with 88 HCCs were treated with (I) PMCT; (II) combined therapy of PEI immediately followed by PMCT; (III) combined therapy of PEI immediately followed by PMCT with occlusion of the feeding artery. The coagulated area was measured at the maximum diameter perpendicular to the needle tract on enhanced computed tomography (CT) scan performed immediately after therapy. The local effect of the treatment was evaluated by enhanced CT follow-up. The rate of complete necrosis was compared in the three treatment groups. RESULTS: The coagulation area and the rate of complete necrosis in group I was 28+/-4.6 mm and 22.5% (7/31), respectively; the coagulation area and complete necrosis in group II were 36+/-8.3 mm and 58.6% (17/29), respectively; the coagulation area and rate of complete necrosis in group III were 46+/-8.5 mm and 92.8% (26/28), respectively. The difference in the coagulation area and the rate of necrosis were significantly larger in the group II than group I (p<0.001, p<0.05), and in group III than group II (p<0.001, p<0.05). CONCLUSIONS: Combined therapy of PEI immediately followed by PMCT, especially with occlusion of the feeding artery can significantly coagulate larger volumes of tumor and improve the rate of complete necrosis.