Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Phosphorylation of OsTGA5 by casein kinase II compromises its suppression of defense-related gene transcription in rice.

Plants manage the high cost of immunity activation by suppressing the expression of defense genes during normal growth and rapidly switching them on upon pathogen invasion. TGAs are key transcription factors controlling the expression of defense genes. However, how TGAs function, especially in monocot plants like rice with continuously high levels of endogenous salicylic acid (SA) remains elusive. In this study, we characterized the role of OsTGA5 as a negative regulator of rice resistance against blast fungus by transcriptionally repressing the expression of various defense-related genes. Moreover, OsTGA5 repressed PTI responses and the accumulation of endogenous SA. Importantly, we showed that the nucleus-localized casein kinase II (CK2) complex interacts with and phosphorylates OsTGA5 on Ser-32, which reduces the affinity of OsTGA5 for the JIOsPR10 promoter, thereby alleviating the repression of JIOsPR10 transcription and increasing rice resistance. Furthermore, the in vivo phosphorylation of OsTGA5 Ser-32 was enhanced by blast fungus infection. The CK2 α subunit, depending on its kinase activity, positively regulated rice defense against blast fungus. Taken together, our results provide a mechanism for the role of OsTGA5 in negatively regulating the transcription of defense-related genes in rice and the repressive switch imposed by nuclear CK2-mediated phosphorylation during blast fungus invasion.

Two-Phase Fermentation Systems for Microbial Production of Plant-Derived Terpenes.

Microbial cell factories, renowned for their economic and environmental benefits, have emerged as a key trend in academic and industrial areas, particularly in the fermentation of natural compounds. Among these, plant-derived terpenes stand out as a significant class of bioactive natural products. The large-scale production of such terpenes, exemplified by artemisinic acid-a crucial precursor to artemisinin-is now feasible through microbial cell factories. In the fermentation of terpenes, two-phase fermentation technology has been widely applied due to its unique advantages. It facilitates in situ product extraction or adsorption, effectively mitigating the detrimental impact of product accumulation on microbial cells, thereby significantly bolstering the efficiency of microbial production of plant-derived terpenes. This paper reviews the latest developments in two-phase fermentation system applications, focusing on microbial fermentation of plant-derived terpenes. It also discusses the mechanisms influencing microbial biosynthesis of terpenes. Moreover, we introduce some new two-phase fermentation techniques, currently unexplored in terpene fermentation, with the aim of providing more thoughts and explorations on the future applications of two-phase fermentation technology. Lastly, we discuss several challenges in the industrial application of two-phase fermentation systems, especially in downstream processing.

A New Quinazolinone Alkaloid along with Known Compounds with Seed-Germination-Promoting Activity from Rhodiola tibetica Endophytic Fungus Penicillium sp. HJT-A-6.

A new quinazolinone alkaloid named peniquinazolinone A (1), as well as eleven known compounds, 2-(2-hydroxy-3-phenylpropionamido)-N-methylbenzamide (2), viridicatin (3), viridicatol (4), (±)-cyclopeptin (5a/5b), dehydrocyclopeptin (6), cyclopenin (7), cyclopenol (8), methyl-indole-3-carboxylate (9), 2,5-dihydroxyphenyl acetate (10), methyl m-hydroxyphenylacetate (11), and conidiogenone B (12), were isolated from the endophytic Penicillium sp. HJT-A-6. The chemical structures of all the compounds were elucidated by comprehensive spectroscopic analysis, including 1D and 2D NMR and HRESIMS. The absolute configuration at C-13 of peniquinazolinone A (1) was established by applying the modified Mosher's method. Compounds 2, 3, and 7 exhibited an optimal promoting effect on the seed germination of Rhodiola tibetica at a concentration of 0.01 mg/mL, while the optimal concentration for compounds 4 and 9 to promote Rhodiola tibetica seed germination was 0.001 mg/mL. Compound 12 showed optimal seed-germination-promoting activity at a concentration of 0.1 mg/mL. Compared with the positive drug 6-benzyladenine (6-BA), compounds 2, 3, 4, 7, 9, and 12 could extend the seed germination period of Rhodiola tibetica up to the 11th day.

Phytochemical, Chemotaxonomic and Bioinformatics Study on Laportea bulbifera (Urticaceae).

Phytochemical investigation of the aerial part of Laportea bulbifera (Siebold & Zucc.) Wedd. (L. bulbifera) showed the isolation of seventeen compounds, including five flavonoids (1-4 and 6), one terpenoid (5), five phenolic acids (7-11), one coumarin (12), two steroids (13-14), and three alkaloids (15-17). Structure elucidations of these compounds were performed on the basis of extensive NMR experiments and compared with the published data in the references. It is remarkable that compounds (3-5) were firstly isolated from the Urticaceae family, compounds (3-8, 11 and 15-17) were firstly obtained from genus Laportea. Furthermore, the result of the chemotaxonomic significance discussion showed that compounds (2-4) may can be served as compound fingerprints to distinguish between species of L. bulbifera and genus Urtica, and what' more, we proposed a bold conjecture that isoflavones can distinguish between species of L. bulbifera and genus Urtica. At the same time, the molecular docking method was used to evaluate the inhibitory effect of these compounds on human steroid 5α-reductase 2 (SRD5α2). The results showed that compounds (1-4 and 6) had better expected effects than the positive drug finasteride can by effectively binding to the active sites of SRD5α2. This study assisted in the future phytochemical and chemotaxonomic research on genus Laportea. Simultaneously, this research provided the theoretical evidence for the application of L. bulbifera in treating benign prostatic hyperplasia (BPH).

RNA Interference-Based Screen Reveals Concerted Functions of MEKK2 and CRCK3 in Plant Cell Death Regulation.

A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by virus-induced gene silencing resembles the mekk1 mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the transcripts of CRCK3 were up-regulated in mekk1, mkk1/2, and mpk4 MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.

Polyketone metabolites isolated from Rhodiola tibetica endohytic fungus Alternaria sp. HJT-Y7 and their SARS-CoV-2 virus inhibitory activitives.

Six new polyketone metabolites, compounds (1-6) and seven known polyketone compounds (7-13) were isolated from Rhodiola tibetica endophytic fungus Alternaria sp. The structural elucidation of five new polyketone metabolites were elucidated on the basis of spectroscopic including 2D NMR and HRMS and spectrometric analysis. Inhibition rate evaluation revealed that compounds 1(EC50 = 0.02 mM), 3(EC50 = 0.3 mM), 6(EC50 = 0.07 mM), 8(EC50 = 0.1 mM) and 9(EC50 = 0.04 mM) had inhibitory effect on the SARS-CoV-2 virus.

Two isopentenyl resorcinols from Peperomia tetraphylla.

Two isopentenyl resorcinols, peperobtusin B and peperobtusin C, have been isolated from Peperomia tetraphylla. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-TOF-MS. Two compounds were evaluated for cytostatic activity against G2, A 549, Hela and HCT 116 cells, but cytostatic activity of both compounds is weak.

Differential Regulation of Two-Tiered Plant Immunity and Sexual Reproduction by ANXUR Receptor-Like Kinases.

Plants have evolved two tiers of immune receptors to detect infections: cell surface-resident pattern recognition receptors (PRRs) that sense microbial signatures and intracellular nucleotide binding domain leucine-rich repeat (NLR) proteins that recognize pathogen effectors. How PRRs and NLRs interconnect and activate the specific and overlapping plant immune responses remains elusive. A genetic screen for components controlling plant immunity identified ANXUR1 (ANX1), a malectin-like domain-containing receptor-like kinase, together with its homolog ANX2, as important negative regulators of both PRR- and NLR-mediated immunity in Arabidopsis thaliana ANX1 constitutively associates with the bacterial flagellin receptor FLAGELLIN-SENSING2 (FLS2) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). Perception of flagellin by FLS2 promotes ANX1 association with BAK1, thereby interfering with FLS2-BAK1 complex formation to attenuate PRR signaling. In addition, ANX1 complexes with the NLR proteins RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2) and RESISTANCE TO P. SYRINGAE PV MACULICOLA1. ANX1 promotes RPS2 degradation and attenuates RPS2-mediated cell death. Surprisingly, a mutation that affects ANX1 function in plant immunity does not disrupt its function in controlling pollen tube growth during fertilization. Our study thus reveals a molecular link between PRR and NLR protein complexes that both associate with cell surface-resident ANX1 and uncovers uncoupled functions of ANX1 and ANX2 during plant immunity and sexual reproduction.

Plant immune signaling: Advancing on two frontiers.

Plants have evolved multiple defense strategies to cope with pathogens, among which plant immune signaling that relies on cell-surface localized and intracellular receptors takes fundamental roles. Exciting breakthroughs were made recently on the signaling mechanisms of pattern recognition receptors (PRRs) and intracellular nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs). This review summarizes the current view of PRRs activation, emphasizing the most recent discoveries about PRRs' dynamic regulation and signaling mechanisms directly leading to downstream molecular events including mitogen-activated protein kinase (MAPK) activation and calcium (Ca2+ ) burst. Plants also have evolved intracellular NLRs to perceive the presence of specific pathogen effectors and trigger more robust immune responses. We also discuss the current understanding of the mechanisms of NLR activation, which has been greatly advanced by recent breakthroughs including structures of the first full-length plant NLR complex, findings of NLR sensor-helper pairs and novel biochemical activity of Toll/interleukin-1 receptor (TIR) domain.

[Preliminary study of dihydroartemisin in inhibiting invasion and metastasis of hepatoma cells].

It is reported that dihydroartemisinin could reduce the expression of phosphorylated adhesion kinase and matrix metalloproteinase-2, inhibit the growth, migration and invasion of ovarian cancer cells, promote the formation of Treg cells through TGF-beta/Smad signaling pathway, and play an immunosuppressive role; dihydroartemisinin could also inhibit the growth of lung cancer cells by inhibiting the expression of vascular endothelial growth factor(VEGF) receptor KDR. However, there are few studies on dihydroartemisinin in hepatocellular carcinoma cells. In order to preliminarily explore the effect of dihydroartemisinin on invasion and metastasis of hepatocellular carcinoma cells, CCK-8 method and crystal violet staining were used to detect the effect of dihydroartemisinin on the growth of hepatocellular carcinoma cell 7402 and highly metastatic hepatocellular carcinoma cell MHCC97 H. The effects of dihydroartemisinin on the invasion and metastasis of hepatocellular carcinoma cell 7402 and highly metastatic hepatocellular carcinoma cell MHCC97 H were studied by using cell wound healing and Transwell. Western blot was used to detect the protein expression of epidermal growth factor receptor(EGFR) and its downstream signaling pathway in cells treated with dihydroartemisinin for 48 hours. The results showed that dihydroartemisinin could inhibit the growth of hepatocellular carcinoma cell 7402 and highly metastatic hepatocellular carcinoma cell MHCC97 H at 25 µmol·L~(-1). As compared with the control group, the number of cell clones was significantly reduced, and the ability of cell migration and invasion was weakened. Western blot results showed that as compared with the control group, dihydroartemisinin group could down-regulate the protein expression of EGFR and its downstream signaling pathways p-AKT, p-ERK, N-cadherin, Snail and Slug, and up-regulate the expression of E-cadherin protein, thus affecting the migration, invasion and metastasis of hepatocellular carcinoma cells 7402 and MHCC97 H.

Correction: Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses.

[This corrects the article DOI: 10.1371/journal.pgen.1004936.].

Two new secolignans from the roots of Urtica fissa.

Two new secolignans, 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxy-3- methoxyphenyl)methyl]butyrolactone (1) and 3,4-trans-3-hydroxymethyl-4- [bis(3,4-dimethoxyphenyl)methyl]butyrolactone (2) have been isolated from the roots of Urtica fissa E.Pritz. Their structures were determined on the basis of spectroscopic methods, especially 1H NMR, 13C NMR, 2D NMR, and HR-ESI-MS. The inhibitory effects on N1 and N2, two subtypes of neuraminidases (NAs), of these two compounds were assayed.

Mechanism of plant immune activation and signaling: Insight from the first solved plant resistosome structure.

This commentary introduces an exciting breakthrough: the first solved structure of a plant NLR immune receptor complex. The significance of this work, including the similarity between the activation of NLRs from plants and animals, and potentially novel mechanism of immune signaling in plants, were discussed and put into perspective.

A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death.

Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses.

PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity.

Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead-associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis, respectively.

Protein poly(ADP-ribosyl)ation regulates arabidopsis immune gene expression and defense responses.

Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks.

Triterpenoids from Euphorbia maculata and Their Anti-Inflammatory Effects.

Euphorbia maculata is a medicinal plant of the Euphorbiaceae family, which can produce anti-inflammatory and cancer chemopreventive agents of triterpenoids. The present study reports on the bioactive triterpenoids of this plant. Two new lanostane-type triterpenoids, named (3S,4S,7S,9R)-4-methyl-3,7-dihydroxy-7(8→9) abeo-lanost-24(28)-en-8-one (1) and 24-hydroperoxylanost-7,25-dien-3ß-ol (2), together with 15 known triterpene derivatives, were isolated from Euphorbia maculata. The structures of the new compounds were determined on the basis of extensive spectroscopic data (UV, MS, ¹H and 13C-NMR, and 2D NMR) analysis. All tetracyclic triterpenoids (1⁻11) were evaluated for their anti-inflammatory effects in the test of TPA-induced inflammation (1 µg/ear) in mice. The triterpenes exhibited significant anti-inflammatory activities.

Three new amino acid derivatives from edible mushroom Pleurotus ostreatus.

Three new amino acid derivatives, oxalamido-L-phenylalanine methyl ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome hydrolyzate (3), together with nine known compounds (4-12), were isolated from the solid culture of edible mushroom Pleurotus ostreatus. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute configurations of 1 and 2 were established by the chiral synthesis and confirmed by circular dichroism (CD) analysis of their total synthesis products and natural isolates. All new compounds were evaluated for their antioxidant effects, antimicrobial activities, and cytotoxic activity. Compounds 1-3 showed weak antifungal activities against Candida albicans with minimum inhibitory concentration (MIC) value of 500 µg/ml.

Polyketide butenolide, diphenyl ether, and benzophenone derivatives from the fungus Aspergillus flavipes PJ03-11.

Five new polyketides including three new butenolides (1-3), one new diphenyl ether (4), and one new benzophenone (5), together with eleven known compounds (6-16) were isolated from a wetland fungus Aspergillus flavipes PJ03-11. Their structures were elucidated on the basis of detailed spectroscopic analysis. All the isolated compounds were tested for their α-glucosidase inhibitory activities. The results showed that compounds 1-3, 6, 7, 11, 15 and 16 exhibited stronger inhibitory activities than acarbose. And the preliminary structure-activity relationships of aspulvinone and diphenyl ether compounds on the α-glucosidase inhibitory activity were reported.