Clathrin Light Chains negatively regulate plant immunity by hijacking the autophagy pathway.

The crosstalk between clathrin-mediated endocytosis (CME) and autophagy pathway has been reported in mammals. However, the interconnection of CME with autophagy has not been established in plants. In this report, we showed that Arabidopsis CLATHRIN LIGHT CHAIN (CLC) subunit 2 and 3 double mutant, clc2-1 clc3-1, phenocopied the Arabidopsis AUTOPHAGY-RELATED GENE (ATG) mutants both in auto-immunity and nutrient sensitivity. Accordingly, the autophagy pathway was significantly compromised in the clc2-1 clc3-1 mutant. Interestingly, we demonstrated with multiple assays that CLC2 directly interacted with ATG8h/ATG8i in a domain-specific manner. As expected, both GFP-ATG8h/GFP-ATG8i and CLC2-GFP were subjected to autophagic degradation and the degradation of GFP-ATG8h was significantly reduced in the clc2-1 clc3-1 mutant. Notably, simultaneously knocking out ATG8h and ATG8i by the CRISPR/CAS9 resulted in an enhanced resistance against Golovinomyces cichoracearum, supporting the functional relevance of the CLC2-ATG8h/8i interactions. In conclusion, our results uncovered a link between the function of CLCs and the autophagy pathway in Arabidopsis.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects.

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5-7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Knocking out NtSARD1a/1b/1c/1d by CRISPR/CAS9 technology reduces the biosynthesis of salicylic acid (SA) and compromises immunity in tetraploid Nicotiana tabacum.

Salicylic acid (SA) is a key phyto-hormone that is essential for plant immunity. SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1), a member of the CBP60 (CALMODULIN-BINDING PROTEIN60) gene family, is one of the major transcription factors regulating the expression of the genes in SA biosynthesis. SARD1 has been extensively studied in model plant Arabidopsis. However, the function of SARD1 homologues in SA biosynthesis and immune responses have rarely been investigated in other plant species. In this study, the CRISPR/CAS9 (Clustered Regularly Interspersed Short Palindromic Repeats/CAS9) technology was used in creating transgenic tobacco mutant lines with 6-8 alleles of four NtSARD1 homologous genes (NtSARD1a/1b/1c/1d) knocked out. No significant difference in morphological phenotype was observed between the transgenic knockout lines and the wild type tobacco plants, indicating that knocking out NtSARD1s does not affect the growth and development in tobacco. However, knocking out or partially knocking out of NtSARD1a/b/c/d resulted in a significantly reduced expression of NtICS1, the key gene in SA biosynthesis pathway, and thus the subsequently decreased SA/SAG accumulations in response to Pst DC3000 (Pseudomonas syrangae pv.tomato DC3000) infection, indicating a key role of NtSARD1 genes in SA biosynthesis in tobacco. As a consequence of reduced SA/SAG accumulation, the Pst DC3000-induced expression of NtPR genes as well as the resistance to Pst DC3000 were both significantly reduced in these knockout lines compared with the wild type tobacco plants. Interestingly, the reductions in the SA/SAG level, NtPR gene induction and Pst DC3000 resistance were positively correlated with the number of alleles being knocked out. Furthermore, LUC reporter gene driven by the promoter of NtICS1 containing two G(A/T)AATT(T/G) motifs could be activated by NtSARD1a, suggesting that NtSARD1a could bind to the core G(A/T)AATT(T/G) motifs and thus activate the expression of LUC reporter. Taken together, our results demonstrated that the NtSARD1 proteins play essential roles in SA biosynthesis and immune responses in tobacco. Our results also demonstrated that the CRISPR/CAS9 technology can overcome gene redundancy and is a powerful tool to study gene functions in polyploid plant species.

De Novo Pterostilbene Production from Glucose Using Modular Coculture Engineering in Escherichia coli.

Pterostilbene, a derivative of resveratrol, is of increasing interest due to its increased bioavailability and potential health benefits. Sustainable production of pterostilbene is important, especially given the challenges of traditional plant extraction and chemical synthesis methods. While engineered microbial cell factories provide a potential alternative for pterostilbene production, most approaches necessitate feeding intermediate compounds. To address these limitations, we adopted a modular coculture engineering strategy, dividing the pterostilbene biosynthetic pathway between two engineered E. coli strains. Using a combination of gene knockout, atmospheric and room-temperature plasma mutagenesis, and error-prone PCR-based whole genome shuffling to engineer strains for the coculture system, we achieved a pterostilbene production titer of 134.84 ± 9.28 mg/L from glucose using a 1:3 inoculation ratio and 0.1% dimethyl sulfoxide supplementation. This represents the highest reported de novo production titer. Our results underscore the potential of coculture systems and metabolic balance in microbial biosynthesis.

Silencing GmATG7 Leads to Accelerated Senescence and Enhanced Disease Resistance in Soybean.

Autophagy plays a critical role in nutrient recycling/re-utilizing under nutrient deprivation conditions. However, the role of autophagy in soybeans has not been intensively investigated. In this study, the Autophay-related gene 7 (ATG7) gene in soybeans (referred to as GmATG7) was silenced using a virus-induced gene silencing approach mediated by Bean pod mottle virus (BPMV). Our results showed that ATG8 proteins were highly accumulated in the dark-treated leaves of the GmATG7-silenced plants relative to the vector control leaves (BPMV-0), which is indicative of an impaired autophagy pathway. Consistent with the impaired autophagy, the dark-treated GmATG7-silenced leaves displayed an accelerated senescence phenotype, which was not seen on the dark-treated BPMV-0 leaves. In addition, the accumulation levels of both H2O2 and salicylic acid (SA) were significantly induced in the GmATG7-silenced plants compared with the BPMV-0 plants, indicating an activated immunity. Consistently, the GmATG7-silenced plants were more resistant against both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV) compared with the BPMV-0 plants. However, the activated immunity in the GmATG7-silenced plant was not dependent upon the activation of MPK3/MPK6. Collectively, our results demonstrated that the function of GmATG7 is indispensable for autophagy in soybeans, and the activated immunity in the GmATG7-silenced plant is a result of impaired autophagy.

Soybean GmSAUL1, a Bona Fide U-Box E3 Ligase, Negatively Regulates Immunity Likely through Repressing the Activation of GmMPK3.

E3 ubiquitin ligases play important roles in plant immunity, but their role in soybean has not been investigated previously. Here, we used Bean pod mottle virus (BPMV)-mediated virus-induced gene silencing (VIGS) to investigate the function of GmSAUL1 (Senescence-Associated E3 Ubiquitin Ligase 1) homologs in soybean. When two closely related SAUL1 homologs were silenced simultaneously, the soybean plants displayed autoimmune phenotypes, which were significantly alleviated by high temperature, suggesting that GmSAUL1a/1b might be guarded by an R protein. Interestingly, silencing GmSAUL1a/1b resulted in the decreased activation of GmMPK6, but increased activation of GmMPK3 in response to flg22, suggesting that the activation of GmMPK3 is most likely responsible for the activated immunity observed in the GmSAUL1a/1b-silenced plants. Furthermore, we provided evidence that GmSAUL1a is a bona fide E3 ligase. Collectively, our results indicated that GmSAUL1 plays a negative role in regulating cell death and immunity in soybean.

GmCBP60b Plays Both Positive and Negative Roles in Plant Immunity.

CBP60b (CALMODULIN-BINDING PROTEIN 60b) is a member of the CBP60 transcription factor family. In Arabidopsis, AtCBP60b not only regulates growth and development but also activates the transcriptions in immune responses. So far, CBP60b has only been studied extensively in the model plant Arabidopsis and rarely in crops. In this study, Bean pod mottle virus (BPMV)-mediated gene silencing (BPMV-VIGS) was used to silence GmCBP60b.1/2 in soybean plants. The silencing of GmCBP60b.1/2 resulted in typical autoimmunity, such as dwarfism and enhanced resistance to both Soybean mosaic virus (SMV) and Pseudomonas syringae pv. glycinea (Psg). To further understand the roles of GmCBP60b in immunity and circumvent the recalcitrance of soybean transformation, we generated transgenic tobacco lines that overexpress GmCBP60b.1. The overexpression of GmCBP60b.1 also resulted in autoimmunity, including spontaneous cell death on the leaves, highly induced expression of PATHOGENESIS-RELATED (PR) genes, significantly elevated accumulation of defense hormone salicylic acid (SA), and significantly enhanced resistance to Pst DC3000 (Pseudomonas syrangae pv. tomato DC3000). The transient coexpression of a luciferase reporter gene driven by the promoter of soybean SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (GmSARD1) (ProGmSARD1::LUC), together with GmCBP60b.1 driven by the 35S promoter, led to the activation of the LUC reporter gene, suggesting that GmCBP60b.1 could bind to the core (A/T)AATT motifs within the promoter region of GmSARD1 and, thus, activate the expression of the LUC reporter. Taken together, our results indicate that GmCBP60b.1/2 play both positive and negative regulatory roles in immune responses. These results also suggest that the function of CBP60b is conserved across plant species.

Silencing GmBIR1 in Soybean Results in Activated Defense Responses.

Receptor-like kinases (RLKs) are a large group of pattern recognition receptors (PRRs) and play a critical role in recognizing pathogens, transducing defense signals, and mediating the activation of immune defense responses. Although extensively studied in the model plant Arabidopsis, studies of RLKs in crops, including soybean, are limited. When a BAK1-interacting receptor-like kinase (BIR1) homolog (referred to as GmBIR1 hereafter) was silenced by the BPMV (Bean pod mottle virus)-induced gene silencing (BPMV-VIGS), it resulted in phenotypes that were reminiscent of constitutively activated defense responses, including a significantly stunted stature with observable cell death on the leaves of the silenced plants. In addition, both SA and H2O2 were over-accumulated in the leaves of the GmBIR1-silenced plants. Consistent with this autoimmune phenotype, GmBIR1-silenced plants exhibited significantly enhanced resistance to both Pseudomonas syringae pv. glycinea (Psg) and Soybean mosaic virus (SMV), two different types of pathogens, compared to the vector control plants. Together, our results indicated that GmBIR1 is a negative regulator of immunity in soybean and the function of BIR1 homologs is conserved in different plant species.

Adaptive laboratory evolution and shuffling of Escherichia coli to enhance its tolerance and production of astaxanthin.

BACKGROUND: Astaxanthin is one of the strongest antioxidants in nature and has been widely used in aquaculture, food, cosmetic and pharmaceutical industries. Numerous stresses caused in the process of a large scale-culture, such as high acetate concentration, high osmolarity, high level of reactive oxygen species, high glucose concentration and acid environment, etc., limit cell growth to reach the real high cell density, thereby affecting astaxanthin production. RESULTS: We developed an adaptive laboratory evolution (ALE) strategy to enhance the production of chemicals by improving strain tolerance against industrial fermentation conditions. This ALE strategy resulted in 18.5% and 53.7% increases in cell growth and astaxanthin production in fed-batch fermentation, respectively. Whole-genome resequencing showed that 65 mutations with amino acid substitution were identified in 61 genes of the shuffled strain Escherichia coli AST-4AS. CRISPR interference (CRISPRi) and activation (CRISPRa) revealed that the shuffled strain with higher astaxanthin production may be associated with the mutations of some stress response protein genes, some fatty acid biosynthetic genes and rppH. Repression of yadC, ygfI and rcsC, activation of rnb, envZ and recC further improved the production of astaxanthin in the shuffled strain E. coli AST-4AS. Simultaneous deletion of yadC and overexpression of rnb increased the production of astaxanthin by 32% in the shuffled strain E. coli AST-4AS. CONCLUSION: This ALE strategy will be powerful in engineering microorganisms for the high-level production of chemicals.

Biosensor-assisted evolution for high-level production of 4-hydroxyphenylacetic acid in Escherichia coli.

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD-aroG, increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future.

Silencing Autophagy-Related Gene 2 (ATG2) Results in Accelerated Senescence and Enhanced Immunity in Soybean.

Autophagy plays a critical role in nutrient recycling and stress adaptations. However, the role of autophagy has not been extensively investigated in crop plants. In this study, soybean autophagy-related gene 2 (GmATG2) was silenced, using virus-induced silencing (VIGS) mediated by Bean pod mottle virus (BPMV). An accelerated senescence phenotype was exclusively observed for the GmATG2-silenced plants under dark conditions. In addition, significantly increased accumulation of both ROS and SA as well as a significantly induced expression of the pathogenesis-related gene 1 (PR1) were also observed on the leaves of the GmATG2-silenced plants, indicating an activated immune response. Consistent with this, GmATG2-silenced plants exhibited a significantly enhanced resistance to Pseudomonas syringae pv. glycinea (Psg) relative to empty vector control plants (BPMV-0). Notably, the activated immunity of the GmATG2-silenced plants was independent of the MAPK signaling pathway. The fact that the accumulation levels of ATG8 protein and poly-ubiquitinated proteins were significantly increased in the dark-treated GmATG2-silenced plants relative to the BPMV-0 plants indicated that the autophagic degradation is compromised in the GmATG2-silenced plants. Together, our results indicated that silencing GmATG2 compromises the autophagy pathway, and the autophagy pathway is conserved in different plant species.

Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering.

Pterostilbene is a derivative of resveratrol with a higher bioavailability and biological activity, which shows antioxidant, anti-inflammatory, antitumor, and antiaging activities. Here, directed evolution and host strain engineering were used to improve the production of pterostilbene in Escherichia coli. First, the heterologous biosynthetic pathway enzymes of pterostilbene, including tyrosine ammonia lyase, p-coumarate: CoA ligase, stilbene synthase, and resveratrol O-methyltransferase, were successively directly evolved through error-prone polymerase chain reaction (PCR). Four mutant enzymes with higher activities of in vivo and in vitro were obtained. The directed evolution of the pathway enzymes increased the pterostilbene production by 13.7-fold. Then, a biosensor-guided genome shuffling strategy was used to improve the availability of the precursor L-tyrosine of the host strain E. coli TYR-30 used for the production of pterostilbene. A shuffled E. coli strain with higher L-tyrosine production was obtained. The shuffled strain harboring the evolved pathway produced 80.04 ± 5.58 mg/l pterostilbene, which is about 2.3-fold the highest titer reported in literatures.

Cell-free Biosynthesis of Chlorogenic Acid Using a Mixture of Chassis Cell Extracts and Purified Spy-Cyclized Enzymes.

A novel cell-free biosynthesis system based on a mixture of chassis cell extracts and purified Spy-cyclized enzymes (CFBS-mixture) was developed. As a demonstration, the CFBS-mixture was applied to chlorogenic acid (CGA) biosynthesis. The mix-and-match and Plackett-Burman experiments demonstrated that Lonicera japonica hydroxycinnamate-CoA quinate transferase and p-hydroxyphenylacetate 3-hydroxylase were the key enzymes for the production of CGA. After optimization of the concentrations of the biosynthetic enzymes in the CFBS-mixture reaction using the Plackett-Burman experimental design and the path of the steepest ascent, 711.26 ± 15.63 mg/L CGA was produced after 16 h, which is 71.1-fold the yield obtained using the conventional crude extract-based CFBS and 9.1-fold the reported yield obtained using the living cells. Based on the CFBS-mixture results, the production of CGA was further enhanced in engineered Escherichia coli. The CFBS-mixture strategy is highly effective and will be useful for high-level CFBS of natural products.

[Consensus on collaborative ethical review of multi-center clinical trials of new drugs of traditional Chinese medicine (version 1.0)].

At present, the issues regarding multi-center clinical trials of new drugs of traditional Chinese medicine(TCM) remain: the lack of agreement on the content and scope of the ethical review among the ethics committee members of the center and the participating units results in repeated review, which leads to a time-consuming ethical review process. Moreover, the review capabilities of the ethics committees of various research centers are uneven, which is not necessarily beneficial to the protection of subjects' rights and safety. In view of the existing problems, to improve the efficiency of ethical review of multi-center clinical trials of new drugs of TCM and avoid repeated reviews, the TCM Clinical Evaluation Professional Committee of Chinese Pharmaceutical Association organized experts to formulate the "Consensus on collaborative ethical review of multi-center clinical trials of new drugs of TCM(version 1.0)"(hereinafter referred to as "Consensus"). The "Consensus" is formulated in accordance with the requirements of relevant documents such as but not limited to "the opinions on deepening the reform of the evaluation and approval system to encourage the innovation of pharmaceutical medical devices", "the regulations of ethical review of biomedical research involving human subjects". The "Consensus" covers the scope of application, formulation principles, conditions for the ethics committee of the center, sharing of ethical review resources, scope and procedure of collaborative review, rights and obligations, etc. The aims of the "Consensus" is to preliminarily explore and establish a scientific and operable ethical review procedure. Additionally, on the basis of fully protecting the rights and interests of the subjects, a collaborative ethical review agreement needs to be signed to clarify the ethical review responsibilities of all parties, to avoid repeated review, and to improve the efficiency and quality of ethical review in multi-center clinical trials of new drugs of TCM.

ATP and NADPH engineering of Escherichia coli to improve the production of 4-hydroxyphenylacetic acid using CRISPRi.

BACKGROUND: 4-Hydroxyphenylacetic acid (4HPAA) is an important raw material for the synthesis of drugs, pesticides and biochemicals. Microbial biotechnology would be an attractive approach for 4HPAA production, and cofactors play an important role in biosynthesis. RESULTS: We developed a novel strategy called cofactor engineering based on clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening (CECRiS) for improving NADPH and/or ATP availability, enhancing the production of 4HPAA. All NADPH-consuming and ATP-consuming enzyme-encoding genes of E. coli were repressed through CRISPRi. After CRISPRi screening, 6 NADPH-consuming and 19 ATP-consuming enzyme-encoding genes were identified. The deletion of the NADPH-consuming enzyme-encoding gene yahK and the ATP-consuming enzyme-encoding gene fecE increased the production of 4HPAA from 6.32 to 7.76 g/L. Automatically downregulating the expression of the pabA gene using the Esa-PesaS quorum-sensing-repressing system further improved the production of 4HPAA. The final strain E. coli 4HPAA-∆yfp produced 28.57 g/L of 4HPAA with a yield of 27.64% (mol/mol) in 2-L bioreactor fed-batch fermentations. The titer and yield are the highest values to date. CONCLUSION: This CECRiS strategy will be useful in engineering microorganisms for the high-level production of bioproducts.

Dual Roles of GSNOR1 in Cell Death and Immunity in Tetraploid Nicotiana tabacum.

S-nitrosoglutathione reductase 1 (GSNOR1) is the key enzyme that regulates cellular homeostasis of S-nitrosylation. Although extensively studied in Arabidopsis, the roles of GSNOR1 in tetraploid Nicotiana species have not been investigated previously. To study the function of NtGSNOR1, we knocked out two NtGSNOR1 genes simultaneously in Nicotiana tabacum using clustered regularly interspaced short palindromic repeats (CRISPR)/caspase 9 (Cas9) technology. To our surprise, spontaneous cell death occurred on the leaves of the CRISPR/Cas9 lines but not on those of the wild-type (WT) plants, suggesting that NtGSNOR1 negatively regulates cell death. The natural cell death on the CRISPR/Cas9 lines could be a result from interactions between overaccumulated nitric oxide (NO) and hydrogen peroxide (H2O2). This spontaneous cell death phenotype was not affected by knocking out two Enhanced disease susceptibility 1 genes (NtEDS11a/1b) and thus was independent of the salicylic acid (SA) pathway. Unexpectedly, we found that the NtGSNOR1a/1b knockout plants displayed a significantly (p < 0.001) enhanced resistance to paraquat-induced cell death compared to WT plants, suggesting that NtGSNOR1 functions as a positive regulator of the paraquat-induced cell death. The increased resistance to the paraquat-induced cell death of the NtGSNOR1a/1b knockout plants was correlated with the reduced level of H2O2 accumulation. Interestingly, whereas the N gene-mediated resistance to Tobacco mosaic virus (TMV) was significantly enhanced (p < 0.001), the resistance to Pseudomonas syringae pv. tomato DC3000 was significantly reduced (p < 0.01) in the NtGSNOR1a/1b knockout lines. In summary, our results indicate that NtGSNOR1 functions as both positive and negative regulator of cell death under different conditions and displays distinct effects on resistance against viral and bacterial pathogens.

Biosensor-Guided Atmospheric and Room-Temperature Plasma Mutagenesis and Shuffling for High-Level Production of Shikimic Acid from Sucrose in Escherichia coli.

Here, we first developed a combined strain improvement strategy of biosensor-guided atmospheric and room-temperature plasma mutagenesis and genome shuffling. Application of this strategy resulted in a 2.7-fold increase in the production of shikimic acid (SA) and a 2.0-fold increase in growth relative to those of the starting strain. Whole-cell resequencing of the shuffled strain and confirmation using CRISPRa/CRISPRi revealed that some membrane protein-related mutant genes are identified as being closely related to the higher SA titer. The engineered shuffling strain produced 18.58 ± 0.56 g/L SA from glucose with a yield of 68% (mol/mol) by fed-batch whole-cell biocatalysis, achieving 79% of the theoretical maximum. Sucrose-utilizing Escherichia coli was engineered for SA production by introducing Mannheimia succiniciproducens ß-fructofuranosidase gene. The resulting sucrose-utilizing E. coli strain produced 24.64 ± 0.32 g/L SA from sucrose with a yield of 1.42 mol/mol by fed-batch whole-cell biocatalysis, achieving 83% of the theoretical maximum.

Emerging Roles of the Selective Autophagy in Plant Immunity and Stress Tolerance.

Autophagy is a conserved recycling system required for cellular homeostasis. Identifications of diverse selective receptors/adaptors that recruit appropriate autophagic cargoes have revealed critical roles of selective autophagy in different biological processes in plants. In this review, we summarize the emerging roles of selective autophagy in both biotic and abiotic stress tolerance and highlight the new features of selective receptors/adaptors and their interactions with both the cargoes and Autophagy-related gene 8s (ATG8s). In addition, we review how the two major degradation systems, namely the ubiquitin-proteasome system (UPS) and selective autophagy, are coordinated to cope with stress in plants. We especially emphasize how plants develop the selective autophagy as a weapon to fight against pathogens and how adapted pathogens have evolved the strategies to counter and/or subvert the immunity mediated by selective autophagy.

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids.

Aromatic compounds derived from aromatic amino acids are an important class of diverse chemicals with a wide range of industrial and commercial applications. They are currently produced via petrochemical processes, which are not sustainable and eco-friendly. In the past decades, significant progress has been made in the construction of microbial cell factories capable of effectively converting renewable carbon sources into value-added aromatics. Here, we systematically and comprehensively review the recent advancements in metabolic engineering and synthetic biology in the microbial production of aromatic amino acid derivatives, stilbenes, and benzylisoquinoline alkaloids. The future outlook concerning the engineering of microbial cell factories for the production of aromatic compounds is also discussed.

Phosphoglycerate Kinase Is Involved in Carbohydrate Utilization, Extracellular Polysaccharide Biosynthesis, and Cell Motility of Xanthomonas axonopodis pv. glycines Independent of Clp.

Phosphoglycerate kinase (Pgk), catalyzing the reversible conversions between glycerate-1.3-2P and glycerate-3P, plays an important role in carbohydrate metabolism. Here, we show that a Pgk-deficient mutant (NΔpgk) of Xanthomonas axonopodis pv. glycines (Xag) could grow in medium with glucose, galactose, fructose, mannose, or sucrose, as the sole carbon source, suggesting that Xag may employ Entner-Doudoroff (ED) and pentose phosphate pathway (PPP), but not glycolysis, to catabolize glucose. NΔpgk could not utilize pyruvate, suggesting that Pgk might be essential for gluconeogenesis. Mutation in pgk led to a reduction of extracellular polysaccharide (EPS) biosynthesis, cell motility, and intracellular ATP. As a result, the virulence of NΔpgk was significantly compromised in soybean. NΔpgk could be fully complemented by the wild-type pgk, but not by clp (encoding Crp-like protein). qRT-PCR analyses demonstrated that pgk is regulated by the HrpG/HrpX cascade, but not by Clp. These results suggest that Pgk is involved in carbohydrate utilization, EPS biosynthesis, and cell motility of Xag independent of Clp.