The Sustained Antidepressant Effects of Ketamine Are Independent of the Lateral Habenula.

Ketamine is known to have a rapid and lasting antidepressant effect. Recent studies have shown that ketamine exerts it rapid antidepressant effect by blocking burst firing in the lateral habenula (LHb). Whether the sustained antidepressant effect of ketamine occurs through the same mechanism has not been explored. Here, using male rats, we found that local infusion of (R,S)-ketamine into the LHb resulted in a rapid antidepressant-like effect 1 h after infusion, which almost returned to baseline levels after 24 h. Intra-LHb injection of (S)-ketamine also showed a significant antidepressant-like effect 1 h after injection, which recovered at 24 h. No significant antidepressant-like effect was found at 1 or 24 h after the administration of (R)-ketamine into the LHb. Injection of (2R,6R)-hydroxynorketamine, a ketamine metabolite, into the LHb did not result in any obvious antidepressant-like effect 1 or 24 h after injection. Systemic administration of (R,S)-ketamine (intraperitoneally) significantly suppressed LHb bursting activity at 1 h, but the inhibitory effect was reversed 24 h after injection. No significant effect of (R,S)-ketamine on miniature excitatory postsynaptic potentials of LHb neurons was found at 1 or 24 h after systemic application. Our study demonstrated that the sustained antidepressant-like effect of ketamine may not depend on burst firing of LHb neurons.SIGNIFICANCE STATEMENT Ketamine exerts it rapid antidepressant effect by blocking burst firing in the lateral habenula (LHb). However, whether the sustained antidepressant effect of ketamine occurs through the same mechanism has not been explored. In the present study, we demonstrated that the sustained antidepressant effect of ketamine may not depend on the burst firing of LHb neurons. This finding may lead to a novel perspective on LHb in the antidepressant effect of ketamine.

Physiological and biochemical response of different resistant alfalfa cultivars against thrips damage.

To investigate physiological and biochemical changes of thrips-resistant alfalfa (Medicago sativa L. cv. Gan-nong No. 9), we aimed at clarifying the response mechanisms of alfalfa against thrips. Medicago sativa L. cv. including thrips-resistant Gan-nong No.9 (G9), thrips-susceptible Gan-nong No.3 (G3) and highly thrips-susceptible WL363HQ (363) were infested with different thrips densities (3, 5, 7 and 9-thrips per branch). The quantitative change in specific nutrients, secondary metabolites, defensive and antioxidant enzymes were measured at seedling stage of the three alfalfa cultivars. The results showed that with the increase of thrips densities, the damage indices, SS, Pro, flavonoids, tannin and H2O2 in G9, G3 and 363 were significantly increased, but PPO and SOD significantly reduced, compared with CK. Furthermore, the tannin and lignin contents of G9 were significantly higher compared to 363, but SP content was significantly lower than G3 and H2O2 content which was further significantly less compared to 363. Correlation analysis observed that the damage index of the three alfalfa cultivars showed a significant positive association with SS, Pro, flavone, tannin, and H2O2 (P < 0.01), while damage index and DW, Chl (a, b, a + b), PPO and SOD showed a significant negative correlation (P < 0.01). Based on principal component comprehensive evaluation, the 5-thrips adults per branch were the critical inoculation threshold for G9 against thrips injury because the score was - 0.048. These results revealed that thrips damage significantly increased the contents of SS, Pro, flavonoids, tannins and H2O2, as well as significantly declined the activities of PPO and SOD in the three cultivars (P < 0.05), moreover, thrips-resistant G9 markedly accumulated lignin content, POD and CAT activity, inhibited Chl (a + b, b) and SP biosynthesis to resist thrips damage.

NF-κB p65-dependent transcriptional regulation of histone deacetylase 2 contributes to the chronic constriction injury-induced neuropathic pain via the microRNA-183/TXNIP/NLRP3 axis.

BACKGROUND: Neuropathic pain is related to the sustained activation of neuroglial cells and the production of proinflammatory cytokines in the spinal dorsal horn. However, the clinical efficacy of currently available treatments is very limited. The transcription factor nuclear factor κB (NF-κB) is a ubiquitously expressed protein family and considered to be crucial in autoimmunity. Thus, our study aimed to examine the influence of NF-κB p65 in chronic constriction injury (CCI)-induced neuropathic pain as well as its underlying mechanism. METHODS: A rat model of neuropathic pain was established by CCI induction followed by isolation of microglial cells. The binding of NF-κB p65 to HDAC2, of miR-183 to TXNIP, and of TXNIP to NLRP3 was investigated. Expression of miR-183, NF-κB p65, HDAC2, TXNIP, and NLRP3 was determined with their functions in CCI rats and microglial cells analyzed by gain- and loss-of-function experiments. RESULTS: NF-κB p65 and HDAC2 were upregulated while miR-183 was downregulated in the dorsal horn of the CCI rat spinal cord. NF-κB p65 was bound to the HDAC2 promoter and then increased its expression. HDAC2 reduced miR-183 expression by deacetylation of histone H4. Additionally, miR-183 negatively regulated TXNIP. Mechanistically, NF-κB p65 downregulated the miR-183 expression via the upregulation of HDAC2 and further induced inflammatory response by activating the TXNIP-NLRP3 inflammasome axis, thus aggravating the neuropathic pain in CCI rats and microglial cells. CONCLUSION: These results revealed a novel transcriptional mechanism of interplay between NF-κB and HDAC2 focusing on neuropathic pain via the miR-183/TXNIP/NLRP3 axis.

Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis.

Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.

Proliferator-Activated Receptor-Gamma Coactivator-1α Haploinsufficiency Promotes Pain Chronification After Burn Injury.

BACKGROUND: Tissue injuries such as surgery and trauma are usually accompanied by simultaneous development of acute pain, which typically resolves along with tissue healing. However, in many cases, acute pain does not resolve despite proper tissue repair; rather, it transitions to chronic pain. In this study, we examined whether proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondria biogenesis, is implicated in pain chronification after burn injury in mice. METHODS: We used PGC-1α and littermates PGC-1α mice of both sex. Burn injury was induced on these mice. Hindpaw mechanical withdrawal thresholds and thermal withdrawal latency were examined. RESULTS: Hindpaw mechanical withdrawal thresholds and thermal withdrawal latencies were comparable at baseline between PGC-1α and PGC-1α mice. After burn injury, both PGC-1α and PGC-1α mice exhibited an initial dramatic decrease of withdrawal parameters at days 3 and 5 after injury. While PGC-1α mice fully recovered their withdrawal parameters to preinjury levels by days 11-14, PGC-1α mice failed to recover those parameters during the same time frame, regardless of sex. Moreover, we found that PGC-1α mice resolved tissue inflammation in a similar fashion to PGC-1α mice using a chemiluminescence-based reactive oxygen species imaging technique. CONCLUSIONS: Taken together, our data suggest that PGC-1α haploinsufficiency promotes pain chronification after burn injury.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

Persistent Nociception Facilitates the Extinction of Morphine-Induced Conditioned Place Preference.

BACKGROUND: As opioid abuse and addiction have developed into a major national health crisis, prescription of opioids for pain management has become more controversial. However, opioids do help some patients by providing pain relief and improving the quality of life. To better understand the addictive properties of opioids under chronic pain conditions, we used a conditioned place preference (CPP) paradigm to examine the rewarding properties of morphine in rats with persistent nociception. METHODS: Spared nerve injury (SNI) model was used to induce persistent nociception in rats. Nociceptive behavior was assessed by von Frey test. CPP test was used to examine the rewarding properties of morphine. RESULTS: Our findings are as follows: (1) SNI rats did not show a difference compared with sham rats in magnitude of morphine-induced CPP 1 day after last morphine injection (2-way analysis of variance; for SNI versus sham, F[1,42] = 0.014, P = .91; and 95% confidence intervals for difference of means, -5.9 [-58 to 46], 0.76 [-51 to 53], and 0.90 [-51 to 53] for 2.5, 5, and 10 mg/kg, respectively); (2) increasing morphine dosage (2.5, 5, and 10 mg/kg) did not further increase the magnitude of CPP in both sham and SNI rats (for dosage: F[2,42] = 0.94, P = .40); and (3) morphine-induced CPP persisted in sham rats but extinguished in SNI rats when tested at 8 days after last morphine injection (for sham versus SNI: Bonferroni correction, P < .006 for both 5 and 10 mg/kg doses; and 95% confidence intervals for difference of means, 80.3 [19.7-141] and 87.0 [26.3-148] for 5 and 10 mg/kg, respectively). CONCLUSIONS: Our data provide new evidence supporting the notion that the brain's reward circuitry changes in the context of persistent pain. This observational study suggests that future investigation into the neurobiology of opioid reward requires consideration of the circumstances in which opioid analgesics are administered.

Exogenous application of salicylic acid improves freezing stress tolerance in alfalfa.

Freezing stress is one of the most detrimental environmental factors that can seriously impact the growth, development, and distribution of alfalfa (Medicago sativa L.). Exogenous salicylic acid (SA) has been revealed as a cost-effective method of improving defense against freezing stress due to its predominant role in biotic and abiotic stress resistance. However, how the molecular mechanisms of SA improve freezing stress resistance in alfalfa is still unclear. Therefore, in this study, we used leaf samples of alfalfa seedlings pretreatment with 200 µM and 0 µM SA, which were exposed to freezing stress (-10°C) for 0, 0.5, 1, and 2h and allowed to recover at normal temperature in a growth chamber for 2 days, after which we detect the changes in the phenotypical, physiological, hormone content, and performed a transcriptome analysis to explain SA influence alfalfa in freezing stress. The results demonstrated that exogenous SA could improve the accumulation of free SA in alfalfa leaves primarily through the phenylalanine ammonia-lyase pathway. Moreover, the results of transcriptome analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway-plant play a critical role in SA alleviating freezing stress. In addition, the weighted gene co-expression network analysis (WGCNA) found that MPK3, MPK9, WRKY22 (downstream target gene of MPK3), and TGACG-binding factor 1 (TGA1) are candidate hub genes involved in freezing stress defense, all of which are involved in the SA signaling pathway. Therefore, we conclude that SA could possibly induce MPK3 to regulate WRKY22 to participate in freezing stress to induced gene expression related to SA signaling pathway (NPR1-dependent pathway and NPR1-independent pathway), including the genes of non-expresser of pathogenesis-related gene 1 (NPR1), TGA1, pathogenesis-related 1 (PR1), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and heat shock protein (HSP). This enhanced the production of antioxidant enzymes such as SOD, POD, and APX, which increases the freezing stress tolerance of alfalfa plants.

Specific interaction based drug loading strategies.

Drug carriers have been commonly used for drug control release, enhancing drug efficacy and/or minimizing side-effects. However, it is still difficult to get a high loading efficiency when encapsulating super hydrophilic drugs with a narrow therapeutic index, such as many neurotoxins. Increasing the carrier proportion can improve drug loading to a certain degree, while the burst released drug when the formulation enters the body may cause overdose side-effects. Moreover, high-dose carriers themselves may increase the metabolic burden of the body. Hence, new drug carriers and/or loading strategies are urgently needed to promote the applications of these drugs. This minireview will introduce drug loading strategies based on specific interactions (between drugs and carriers) and will discuss the challenges and perspectives of these strategies. This work is expected to provide alternative inspiration for the delivery of hydrophilic drugs.

Comparative Transcriptome Analysis Reveals New Insight of Alfalfa (Medicago sativa L.) Cultivars in Response to Abrupt Freezing Stress.

Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa (Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance "Gannong NO.3" and freezing-sensitive "WL326GZ" exposure to -10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in "Gannong NO.3" were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in "WL326GZ" were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca2+ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.

Sp1 Inhibits PGC-1α via HDAC2-Catalyzed Histone Deacetylation in Chronic Constriction Injury-Induced Neuropathic Pain.

BACKGROUND: Our previous study has illuminated that PGC-1α downregulation promoted chronification of pain after burn injury. RNA-seq analysis predicted association between Sp1 and chronic constriction injury (CCI)-provoked neuropathic pain. Further ChIP-Atlas data investigation suggested the binding to Sp1 to PGC-1α. Thereby, we performed this study to illustrate the functional relevance of the Sp1/PGC-1α axis in neuropathic pain. METHODS: Neuropathic pain was induced by CCI in vivo in rats, followed by assessment of neuropathic pain-like behaviors. The expression of Sp1 and correlated genes was determined in CCI rat spinal cord tissues. Furthermore, microglia were exposed to lipopolysaccharide (LPS) to mimic inflammation and then cocultured with neurons. Knockdown and ectopic expression methods were used in vivo and in vitro to define the role the Sp1/HDAC2/PGC-1α axis. RESULTS: Sp1 expression was upregulated in spinal cord tissues of CCI rats. Silencing Sp1 ameliorated CCI-induced neuropathic pain, as reflected by elevated paw withdrawal threshold and paw withdrawal latency, as well as alleviated microglia activation, neuronal dysfunction, inflammatory responses, mitochondrial dysfunction, and oxidative stress in spinal cord tissues. Sp1 knockdown also reversed LPS-induced microglial inflammation and neuronal dysfunction. Sp1 promoted histone deacetylation in the PGC-1α promoter and inhibited PGC-1α expression via recruiting HDAC2. PGC-1α overexpression diminished CCI-induced neuropathic pain and LPS-induced inflammation and mitochondrial dysfunction, based on which Sp1 aggravated microglial inflammation and neuronal dysfunction in neuropathic pain. CONCLUSION: This study elucidated the promoting effects of Sp1 on CCI-induced neuropathic pain via the HDAC2/PGC-1α axis.

Mitophagy impairment is involved in sevoflurane-induced cognitive dysfunction in aged rats.

Postoperative cognitive dysfunction (POCD) is frequently observed in elderly patients following anesthesia, but its pathophysiological mechanisms have not been fully elucidated. Sevoflurane was reported to repress autophagy in aged rat neurons; however, the role of mitophagy, which is crucial for the control of mitochondrial quality and neuronal health, in sevoflurane-induced POCD in aged rats remains undetermined. Therefore, this study investigated whether mitophagy impairment is involved in sevoflurane-induced cognitive dysfunction. We found sevoflurane treatment inhibited mitochondrial respiration and mitophagic flux, changes in mitochondria morphology, impaired lysosomal acidification, and increased Tomm20 and deceased LAMP1 accumulation were observed in H4 cell and aged rat models. Rapamycin counteracted ROS induced by sevoflurane, restored mitophagy and improved mitochondrial function. Furthermore, rapamycin ameliorated the cognitive deficits observed in aged rats given sevoflurane anesthesia as determined by the Morris water maze test; this improvement was associated with an increased number of dendritic spines and pyramidal neurons. Overexpression of PARK2, but not mutant PARK2 lacking enzyme activity, in H4 cells decreased ROS and Tomm20 accumulation and reversed mitophagy dysfunction after sevoflurane treatment. These findings suggest that mitophagy dysfunction could be a mechanism underlying sevoflurane-induced POCD and that activating mitophagy may provide a new strategy to rescue cognitive deficits.

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.

Genomic features of bacterial adaptation to plants.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes.

Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.

SacB-SacR Gene Cassette As the Negative Selection Marker to Suppress Agrobacterium Overgrowth in Agrobacterium-Mediated Plant Transformation.

Agrobacterium overgrowth is a common problem in Agrobacterium-mediated plant transformation. To suppress the Agrobacterium overgrowth, various antibiotics have been used during plant tissue culture steps. The antibiotics are expensive and may adversely affect plant cell differentiation and reduce plant transformation efficiency. The SacB-SacR proteins are toxic to most Agrobacterium tumefaciens strains when they are grown on culture medium supplemented with sucrose. Therefore, SacB-SacR genes can be used as negative selection markers to suppress the overgrowth of A. tumefaciens in the plant tissue culture process. We generated a mutant A. tumefaciens strain GV2260 (recA-SacB/R) that has the SacB-SacR cassette inserted into the bacterial genome at the recA gene locus. The mutant Agrobacterium strain is sensitive to sucrose but maintains its ability to transform plant cells in both transient and stable transformation assays. We demonstrated that the mutant strain GV2260 (recA-SacB/R) can be inhibited by sucrose that reduces the overgrowth of Agrobacterium and therefore improves the plant transformation efficiency. We employed GV2260 (recA-SacB/R) to generate stable transgenic N. benthamiana plants expressing a CRISPR-Cas9 for knocking out a WRKY transcription factor.

Crystal Structure of Xanthomonas AvrRxo1-ORF1, a Type III Effector with a Polynucleotide Kinase Domain, and Its Interactor AvrRxo1-ORF2.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) disease on rice plants. Xoc delivers a type III effector AvrRxo1-ORF1 into rice plant cells that can be recognized by disease resistance (R) protein Rxo1, and triggers resistance to BLS disease. However, the mechanism and virulence role of AvrRxo1 is not known. In the genome of Xoc, AvrRxo1-ORF1 is adjacent to another gene AvrRxo1-ORF2, which was predicted to encode a molecular chaperone of AvrRxo1-ORF1. We report the co-purification and crystallization of the AvrRxo1-ORF1:AvrRxo1-ORF2 tetramer complex at 1.64 Å resolution. AvrRxo1-ORF1 has a T4 polynucleotide kinase domain, and expression of AvrRxo1-ORF1 suppresses bacterial growth in a manner dependent on the kinase motif. Although AvrRxo1-ORF2 binds AvrRxo1-ORF1, it is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain. AvrRxo1-ORF2 functions to suppress the bacteriostatic activity of AvrRxo1-ORF1 in bacterial cells.