Loop-mediated isothermal amplification (LAMP)/Cas12a assay for detection of Ralstonia solanacearum in tomato.

Introduction: Bacterial wilt (BW) caused by the aerobic, Gram-negative pathogenic species Ralstonia solanacearum (RS) is a major disease impacting commercial agriculture worldwide. Asian phylotype I of RS is the cause of tomato bacterial wilt, which has caused severe economic losses in southern China for many years. An urgent priority in control of bacterial wilt is development of rapid, sensitive, effective methods for detection of RS. Methods: We describe here a novel RS detection assay based on combination of loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a. crRNA1, with high trans-cleavage activity targeting hrpB gene, was selected out of four candidate crRNAs. Two visual detection techniques, involving naked-eye observation of fluorescence and lateral flow strips, were tested and displayed high sensitivity and strong specificity. Results and Discussion: The LAMP/Cas12a assay accurately detected RS phylotype Ⅰ in 14 test strains, and showed low detection limit (2.0 × 100 copies). RS in tomato stem tissue and soil samples from two field sites with suspected BW infection was identified accurately, suggesting potential application of LAMP/Cas12a assay as point-of-care test (POCT). The overall detection process took less than 2 h and did not require professional lab equipment. Our findings, taken together, indicate that LAMP/Cas12a assay can be developed as an effective, inexpensive technique for field detection and monitoring of RS.

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding.

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Phytocytokines function as immunological modulators of plant immunity.

Plant plasma membrane-resident immune receptors regulate plant immunity by recognizing microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and phytocytokines. Phytocytokines are plant endogenous peptides, which are usually produced in the cytosol and released into the apoplast when plant encounters pathogen infections. Phytocytokines regulate plant immunity through activating an overlapping signaling pathway with MAMPs/DAMPs with some unique features. Here, we highlight the current understanding of phytocytokine production, perception and functions in plant immunity, and discuss how plants and pathogens manipulate phytocytokine signaling for their own benefits during the plant-pathogen warfare.

The Arabidopsis MIK2 receptor elicits immunity by sensing a conserved signature from phytocytokines and microbes.

Sessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that the Arabidopsis receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) from Brassicaceae plants as well as proteins present in fungal Fusarium spp. and bacterial Comamonadaceae, and elicits various immune responses. SCOOP signature peptides trigger immune responses and altered root development in a MIK2-dependent manner with a sub-nanomolar sensitivity. SCOOP12 directly binds to the extracellular leucine-rich repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identifies a plant receptor that bears a dual role in sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.

[Analysis of the incidence of syncope and the influencing factors of death in patients with cardiovascular critical emergency].

OBJECTIVE: To observe the incidence of syncope in patients with acute and critical cardiovascular diseases and to explore the risk factors of death. METHODS: 925 cases of acute heart failure, acute myocardial infarction, pulmonary embolism, arrhythmia and aortic dissection rupture who participated in Prospective, Multi-Center Registered Research Project for Chinese Syncope Patients from March 2018 to March 2020, admitted to the department of emergency of Nanyang Second General Hospital were selected as the research objects. The incidence and mortality of syncope were recorded, and the patients were divided into syncope group and non-syncope group according to whether they were accompanied by syncope or not. The incidence of syncope in male and female patients with different cardiovascular critical diseases, the age and mortality of cardiovascular critical patients with syncope or not were analyzed and compared. Multivariate Logistic regression analysis was used to analyze the risk factors of death, and receiver operating characteristic curve (ROC curve) was drawn to evaluate the predictive value of risk factors on the prognosis of patients. RESULTS: The incidence of syncope in 5 kinds of cardiovascular critical patients from high to low was: acute myocardial infarction 3.03% (28/925), arrhythmia 2.70% (25/925), pulmonary embolism 1.51% (14/925), aortic dissection rupture 1.41% (13/925), acute heart failure 0.65% (6/925), with statistically significant differences (χ2 = 10.765, P = 0.010). There was no significant difference in the incidence of syncope between male and female patients with pulmonary embolism, aortic dissection rupture, acute myocardial infarction, arrhythmia and acute heart failure. The age of patients with aortic dissection rupture, acute myocardial infarction and arrhythmia in syncope group were significantly higher than those in non-syncope group [aortic dissection rupture (years old): 66.29±15.64 vs. 57.63±14.23, acute myocardial infarction (years old): 69.55±15.13 vs. 62.10±15.75, arrhythmia (years old): 70.48±14.93 vs. 60.29±16.31, all P < 0.05]. The mortality of patients with pulmonary embolism, aortic dissection rupture, acute myocardial infarction, arrhythmia, acute heart failure in syncope group were significantly higher than those in non-syncope group [pulmonary embolism: 5.81% (5/86) vs. 0.95% (8/839), aortic dissection rupture: 4.65% (4/86) vs. 0.60% (5/839), acute myocardial infarction: 4.65% (4/86) vs. 1.19% (10/839), arrhythmia: 2.33% (2/86) vs. 0.95% (8/839), acute heart failure: 2.33% (2/86) vs. 0.60% (5/839), all P < 0.05]. Multivariate Logistic regression analysis showed that age [odds ratio (OR) = 2.158, 95% confidence interval (95%CI) was 0.921-4.785, P = 0.000], pulmonary embolism (OR = 15.391, 95%CI was 8.904-27.314, P = 0.001), aortic dissection rupture (OR = 13.079, 95%CI was 6.237-25.509, P = 0.000), acute myocardial infarction (OR = 18.826, 95%CI was 10.420-32.921, P = 0.000), syncope (OR = 4.940, 95%CI was 1.764-9.287, P = 0.000) were risk factors for the prognosis of patients with acute and critical cardiovascular diseases. ROC curve analysis showed that syncope had a certain predictive value for 28-day prognosis of patients [the area under the ROC curve (AUC) = 0.760, P = 0.000], when the cut-off value was 4.12, the sensitivity was 88.51%, the specificity was 78.05%, the positive predictive value was 81.31%, and the negative predictive value was 84.27%. CONCLUSIONS: Syncope is an independent risk factor of death in patients with acute and critical cardiovascular diseases. For patients with syncope as the chief complaint, we should quickly identify the types of acute and critical diseases and assess the risk of sudden death.

OsRRM, an RNA-Binding Protein, Modulates Sugar Transport in Rice (Oryza sativa L.).

Sugar allocation between vegetative and reproductive tissues is vital to plant development, and sugar transporters play fundamental roles in this process. Although several transcription factors have been identified that control their transcription levels, the way in which the expression of sugar transporter genes is controlled at the posttranscriptional level is unknown. In this study, we showed that OsRRM, an RNA-binding protein, modulates sugar allocation in tissues on the source-to-sink route. The OsRRM expression pattern partly resembles that of several sugar transporter and transcription factor genes that specifically affect sugar transporter gene expression. The messenger RNA levels of almost all of the sugar transporter genes are severely reduced in the osrrm mutant, and this alters sugar metabolism and sugar signaling, which further affects plant height, flowering time, seed size, and starch synthesis. We further showed that OsRRM binds directly to messenger RNAs encoded by sugar transporter genes and thus may stabilize their transcripts. Therefore, we have uncovered the physiological function of OsRRM, which sheds new light on sugar metabolism and sugar signaling.

Endocytosis of BRASSINOSTEROID INSENSITIVE1 Is Partly Driven by a Canonical Tyr-Based Motif.

Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles via the recognition of motifs based on Tyr or di-Leu in their cytoplasmic tails. However, in plants, very little is known about how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis (Arabidopsis thaliana), the brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) undergoes endocytosis, which depends on clathrin and AP-2. Here, we demonstrate that BRI1 binds directly to the medium AP-2 subunit (AP2M). The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed Tyr-based endocytic motifs. The Tyr-to-Phe substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2-dependent internalization of PM proteins via the recognition of functional Tyr motifs also operates in plants.

The malectin-like receptor-like kinase LETUM1 modulates NLR protein SUMM2 activation via MEKK2 scaffolding.

The innate immune system detects pathogen-derived molecules via specialized immune receptors to prevent infections1-3. Plant immune receptors include cell surface-resident pattern recognition receptors (PRRs, including receptor-like kinases (RLKs)), and intracellular nucleotide-binding domain leucine-rich repeat proteins (NLRs). It remains enigmatic how RLK- and NLR-mediated signalling are connected. Disruption of an immune-activated MEKK1-MKK1/2-MPK4 MAPK cascade activates the NLR SUMM2 via the MAPK kinase kinase MEKK2, leading to autoimmunity4-9. To gain insights into the mechanisms underlying SUMM2 activation, we used an RNA interference-based genetic screen for mekk1 autoimmune suppressors and identified an uncharacterized malectin-like RLK, named LETUM1 (LET1), as a specific regulator of mekk1-mkk1/2-mpk4 autoimmunity via complexing with both SUMM2 and MEKK2. MEKK2 scaffolds LET1 and SUMM2 for protein stability and association, and counter-regulates the F-box protein CPR1-mediated SUMM2 ubiquitination and degradation, thereby regulating SUMM2 accumulation and activation. Our study indicates that malectin-like RLK LET1 senses the perturbance of cellular homoeostasis caused by the deficiency in immune-activated signalling and activates the NLR SUMM2-mediated autoimmunity via MEKK2 scaffolding.

Brassinosteroids: Multidimensional Regulators of Plant Growth, Development, and Stress Responses.

Brassinosteroids (BRs) are a group of polyhydroxylated plant steroid hormones that are crucial for many aspects of a plant's life. BRs were originally characterized for their function in cell elongation, but it is becoming clear that they play major roles in plant growth, development, and responses to several stresses such as extreme temperatures and drought. A BR signaling pathway from cell surface receptors to central transcription factors has been well characterized. Here, we summarize recent progress toward understanding the BR pathway, including BR perception and the molecular mechanisms of BR signaling. Next, we discuss the roles of BRs in development and stress responses. Finally, we show how knowledge of the BR pathway is being applied to manipulate the growth and stress responses of crops. These studies highlight the complex regulation of BR signaling, multiple points of crosstalk between BRs and other hormones or stress responses, and the finely tuned spatiotemporal regulation of BR signaling.

Plant Growth Promotion Driven by a Novel Caulobacter Strain.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.

Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination.

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase-substrate association regulated by phosphorylation.

Imaging cellulose synthase motility during primary cell wall synthesis in the grass Brachypodium distachyon.

The mechanism of cellulose synthesis has been studied by characterizing the motility of cellulose synthase complexes tagged with a fluorescent protein; however, this approach has been used exclusively on the hypocotyl of Arabidopsis thaliana. Here we characterize cellulose synthase motility in the model grass, Brachypodium distachyon. We generated lines in which mEGFP is fused N-terminal to BdCESA3 or BdCESA6 and which grew indistinguishably from the wild type (Bd21-3) and had dense fluorescent puncta at or near the plasma membrane. Measured with a particle tracking algorithm, the average speed of GFP-BdCESA3 particles in the mesocotyl was 164 ± 78 nm min-1 (error gives standard deviation [SD], n = 1451 particles). Mean speed in the root appeared similar. For comparison, average speed in the A. thaliana hypocotyl expressing GFP-AtCESA6 was 184 ± 86 nm min-1 (n = 2755). For B. distachyon, we quantified root diameter and elongation rate in response to inhibitors of cellulose (dichlorobenylnitrile; DCB), microtubules (oryzalin), or actin (latrunculin B). Neither oryzalin nor latrunculin affected the speed of CESA complexes; whereas, DCB reduced average speed by about 50% in B. distachyon and by about 35% in A. thaliana. Evidently, between these species, CESA motility is well conserved.

Molecular Structure and Physicochemical Properties of Starches from Rice with Different Amylose Contents Resulting from Modification of OsGBSSI Activity.

OsGBSSI, encoded by the Waxy (Wx) gene, is the key enzyme in the synthesis of amylose chains. Transgenic rice lines with various GBSSI activities were previously developed via site-directed mutagenesis of the Wx gene in the glutinous cultivar Guanglingxiangnuo (GLXN). In this study, grain morphology, molecular structure, and physicochemical properties were investigated in four transgenic lines with modified OsGBSSI activity and differences in amylose content. A milky opaque appearance was observed in low- and non-amylose rice grains due to air spaces in the starch granules. Gel permeation chromatography (GPC) and high-performance anion-exchange chromatography (HPAEC) analyses showed that although OsGBSSI can synthesize intermediate and extra-long amylopectin chains, it is mainly responsible for the longer amylose chains. Amylose content was positively correlated with trough viscosity, final viscosity, setback viscosity, pasting time, pasting temperature, and gelatinization temperature and negatively with gel consistency, breakdown viscosity, gelatinization enthalpy, and crystallinity. Overall, the findings suggest that OsGBSSI may be also involved in amylopectin biosynthesis, in turn affecting grain appearance, thermal and pasting properties, and the crystalline structure of starches in the rice endosperm.

Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.).

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.

Oligomerization of rice granule-bound starch synthase 1 modulates its activity regulation.

Granule-bound starch synthase 1 (GBSS1) is responsible for amylose synthesis in cereals, and this enzyme is regulated at the transcriptional and post-transcriptional levels. In this study, we show that GBSS1 from Oryza sativa L. (OsGBSS1) can form oligomers in rice endosperm, and oligomerized OsGBSS1 exhibits much higher specific enzymatic activity than the monomer. A monomer-oligomer transition equilibrium for OsGBSS1 occurs in the endosperm during development. Redox potential is a key factor affecting the oligomer percentage as well as the enzymatic activity of OsGBSS1. Adenosine diphosphate glucose, the direct donor of glucose, also impacts OsGBSS1 oligomerization in a concentration-dependent manner. OsGBSS1 oligomerization is influenced by phosphorylation status, which was strongly enhanced by Mitogen-activated protein kinase (MAPK) and ATP treatment and was sharply weakened by protein phosphatase (PPase) treatment. The activity of OsGBSS1 affects the ratio of amylose to amylopectin and therefore the eating quality of rice. Understanding the regulation of OsGBSS1 activity may lead to the improvement of rice eating quality.

OsRRMh, a Spen-like gene, plays an important role during the vegetative to reproductive transition in rice.

OsRRMh, a homologue of OsRRM, encodes a Spen-like protein, and is composed of two N-terminal RNA recognition motifs (RRM) and one C-terminal Spen paralogue and an orthologue C-terminal domain (SPOC). The gene has been found to be constitutively expressed in the root, stem, leaf, spikelet, and immature seed, and alternative splicing patterns were confirmed in different tissues, which may indicate diverse functions for OsRRMh. The OsRRMh dsRNAi lines exhibited late-flowering and a larger panicle phenotype. When full-length OsRRMh and/or its SPOC domain were overexpressed, the fertility rate and number of spikelets per panicle were both markedly reduced. Also, overexpression of OsRRMh in the Arabidopsis fpa mutant did not restore the normal flowering time, and it delayed flowering in Col plants. Therefore, we propose that OsRRMh may confer one of its functions in the vegetative-to-reproductive transition in rice (Oryza sativa L. subsp. japonica cv. Zhonghua No. 11 (ZH11)).