Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector.

Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.

The OsBDR1-MPK3 module negatively regulates blast resistance by suppressing the jasmonate signaling and terpenoid biosynthesis pathway.

Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae (M. oryzae) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae-induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae-elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic (allene oxide cyclase (AOC)/signaling (MYC2) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae, and their biosynthesis genes (TPS3 and TPS29) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.

Role of miRNAs in glucose metabolism of mouse cumulus cells†.

It is known that the oocyte has a limited capacity to acquire and metabolize glucose, and it must rely on cumulus cells (CCs) to take up glucose and produce pyruvate for use to produce ATP through oxidative phosphorylation. We therefore propose that miRNAs might regulate glucose metabolism (GM) in CCs and might be used as markers for oocyte quality assessment. Here, mouse CC models with impaired glycolysis or pentose phosphate pathway (PPP) were established, and miRNAs targeting the key enzymes in glycolysis/PPP were predicted using the miRNA target prediction databases. Expression of the predicted miRNAs was compared between CCs with normal and impaired glycolysis/PPP to identify candidate miRNAs. Function of the candidate miRNAs was validated by transfecting CCs or cumulus-oocyte-complexes (COCs) with miRNA inhibitors and observing effects on glucose metabolites of CCs and on competence of oocytes. The results validated that miR-23b-3p, let-7b-5p, 34b-5p and 145a-5p inhibited glycolysis, and miR-24-3p, 3078-3p,183-5p and 7001-5p inhibited PPP of CCs. Our observation using a more physiologically relevant model (intact cultured COCs) further validated the four glycolysis-targeting miRNAs we identified. Furthermore, miR-let-7b-5p, 34b-5p and 145a-5p may also inhibit PPP, as they decreased the production of glucose-6-phosphate. In conclusion, miRNAs play critical roles in GM of CCs and may be used as markers for oocyte quality assessment. Summary sentence:  We identified and validated eight new miRNAs that inhibit glycolysis and/or pentose phosphate pathways in cumulus cells (CCs) suggesting that miRNAs play critical roles in glucose metabolism of CCs and may be used for oocyte quality markers.

OsEIL2 balances rice immune responses against (hemi)biotrophic and necrotrophic pathogens via the salicylic acid and jasmonic acid synergism.

Plants typically activate distinct defense pathways against various pathogens. Heightened resistance to one pathogen often coincides with increased susceptibility to another pathogen. However, the underlying molecular basis of this antagonistic response remains unclear. Here, we demonstrate that mutants defective in the transcription factor ETHYLENE-INSENSITIVE 3-LIKE 2 (OsEIL2) exhibited enhanced resistance to the biotrophic bacterial pathogen Xanthomonas oryzae pv oryzae and to the hemibiotrophic fungal pathogen Magnaporthe oryzae, but enhanced susceptibility to the necrotrophic fungal pathogen Rhizoctonia solani. Furthermore, necrotroph-induced OsEIL2 binds to the promoter of OsWRKY67 with high affinity, leading to the upregulation of salicylic acid (SA)/jasmonic acid (JA) pathway genes and increased SA/JA levels, ultimately resulting in enhanced resistance. However, biotroph- and hemibiotroph-induced OsEIL2 targets OsERF083, resulting in the inhibition of SA/JA pathway genes and decreased SA/JA levels, ultimately leading to reduced resistance. Our findings unveil a previously uncharacterized defense mechanism wherein two distinct transcriptional regulatory modules differentially mediate immunity against pathogens with different lifestyles through the transcriptional reprogramming of phytohormone pathway genes.

Influence of tumor thrombus morphology on the surgical complexity in renal cell carcinoma with inferior vena cava tumor thrombus: a single-center, large-sample study from China.

BACKGROUND: The morphology of tumor thrombus varies from person to person and it may affect surgical methods and tumor prognosis. However, studies on the morphology of tumor thrombus are limited. The purpose of our study was to evaluate the impact of tumor thrombus morphology on surgical complexity. METHODS: We retrospectively reviewed the clinical data of 229 patients with renal cell carcinoma combined with inferior vena cava (IVC) tumor thrombus who underwent surgical treatment at Peking University Third Hospital between January 2014 and December 2021. The patients were divided into floating morphology (107 patients) and filled morphology (122 patients) tumor thrombi groups. Chi-square and Mann-Whitney U tests were used for categorical and continuous variables, respectively. Postoperative complications were evaluated using the Clavien-Dindo surgical complication classification method. RESULTS: Patients with filled morphology tumor thrombus required more surgical techniques than those with floating morphology tumor thrombus, which was reflected in more open surgeries (P < 0.001), more IVC interruptions (P <0.001), lesser use of the delayed occlusion of the proximal inferior vena cava (DOPI) technique (P < 0.001), and a greater need for cut-off of the short hepatic vein (P < 0.001) and liver dissociation (P = 0.001). Filled morphology significantly increased the difficulty of surgery in patients with renal cell carcinoma with tumor thrombus, reflected in longer operation time (P < 0.001), more surgical blood loss (P <0.001), more intra-operative blood transfusion (P < 0.001), and longer postoperative hospital stay (P < 0.001). Filled morphology tumor thrombus also led to more postoperative complications (53% vs. 20%; P < 0.001). CONCLUSION: Compared with floating morphology thrombus, filled morphology thrombus significantly increased the difficulty of surgery in patients with renal cell carcinoma with IVC tumor thrombus.

Effects of Maize Chlorotic Mottle Virus and Potyvirus Resistance on Maize Lethal Necrosis Disease.

Maize lethal necrosis (MLN) is a viral disease caused by host co-infection by maize chlorotic mottle virus (MCMV) and a potyvirus, such as sugarcane mosaic virus (SCMV). The disease is most effectively managed by growing MLN-resistant varieties. However, the relative importance of MCMV and potyvirus resistance in managing this synergistic disease is poorly characterized. In this study, we evaluated the effects of SCMV and/or MCMV resistance on disease, virus titers, and synergism and explored expression patterns of known potyvirus resistance genes TrxH and ABP1. MLN disease was significantly lower in both the MCMV-resistant and SCMV-resistant inbred lines compared with the susceptible control Oh28. Prior to 14 days postinoculation (dpi), MCMV titers in resistant lines N211 and KS23-6 were more than 100,000-fold lower than found in the susceptible Oh28. However, despite no visible symptoms, titer differences between MCMV-resistant and -susceptible lines were negligible by 14 dpi. In contrast, systemic SCMV titers in the potyvirus-resistant line, Pa405, ranged from 130,000-fold to 2 million-fold lower than susceptible Oh28 as disease progressed. Initial TrxH expression was up to 49,000-fold lower in Oh28 compared with other genotypes, whereas expression of ABP1 was up to 4.5-fold lower. Measures of virus synergy indicate that whereas MCMV resistance is effective in early infection, strong potyvirus resistance is critical for reducing synergist effects of co-infection on MCMV titer. These results emphasize the importance of both potyvirus resistance and MCMV resistance in an effective breeding program for MLN management.

APIP5 functions as a transcription factor and an RNA-binding protein to modulate cell death and immunity in rice.

Many transcription factors (TFs) in animals bind to both DNA and mRNA, regulating transcription and mRNA turnover. However, whether plant TFs function at both the transcriptional and post-transcriptional levels remains unknown. The rice (Oryza sativa) bZIP TF AVRPIZ-T-INTERACTING PROTEIN 5 (APIP5) negatively regulates programmed cell death and blast resistance and is targeted by the effector AvrPiz-t of the blast fungus Magnaporthe oryzae. We demonstrate that the nuclear localization signal of APIP5 is essential for APIP5-mediated suppression of cell death and blast resistance. APIP5 directly targets two genes that positively regulate blast resistance: the cell wall-associated kinase gene OsWAK5 and the cytochrome P450 gene CYP72A1. APIP5 inhibits OsWAK5 expression and thus limits lignin accumulation; moreover, APIP5 inhibits CYP72A1 expression and thus limits reactive oxygen species production and defense compounds accumulation. Remarkably, APIP5 acts as an RNA-binding protein to regulate mRNA turnover of the cell death- and defense-related genes OsLSD1 and OsRac1. Therefore, APIP5 plays dual roles, acting as TF to regulate gene expression in the nucleus and as an RNA-binding protein to regulate mRNA turnover in the cytoplasm, a previously unidentified regulatory mechanism of plant TFs at the transcriptional and post-transcriptional levels.

Analysis of risk factors, pathogenic bacteria characteristics, and drug resistance of postoperative surgical site infection in adults with limb fractures.

PURPOSE: We carried out the study aiming to explore and analyze the risk factors, the distribution of pathogenic bacteria, and their antibiotic-resistance characteristics influencing the occurrence of surgical site infection (SSI), to provide valuable assistance for reducing the incidence of SSI after traumatic fracture surgery. METHODS: A retrospective case-control study enrolling 3978 participants from January 2015 to December 2019 receiving surgical treatment for traumatic fractures was conducted at Tangdu Hospital of Air Force Medical University. Baseline data, demographic characteristics, lifestyles, variables related to surgical treatment, and pathogen culture were harvested and analyzed. Univariate analyses and multivariate logistic regression analyses were used to reveal the independent risk factors of SSI. A bacterial distribution histogram and drug-sensitive heat map were drawn to describe the pathogenic characteristics. RESULTS: Included 3978 patients 138 of them developed SSI with an incidence rate of 3.47% postoperatively. By logistic regression analysis, we found that variables such as gender (males) (odds ratio (OR) = 2.012, 95% confidence interval (CI): 1.235 - 3.278, p = 0.005), diabetes mellitus (OR = 5.848, 95% CI: 3.513 - 9.736, p < 0.001), hypoproteinemia (OR = 3.400, 95% CI: 1.280 - 9.031, p = 0.014), underlying disease (OR = 5.398, 95% CI: 2.343 - 12.438, p < 0.001), hormonotherapy (OR = 11.718, 95% CI: 6.269 - 21.903, p < 0.001), open fracture (OR = 29.377, 95% CI: 9.944 - 86.784, p < 0.001), and intraoperative transfusion (OR = 2.664, 95% CI: 1.572 - 4.515, p < 0.001) were independent risk factors for SSI, while, aged over 59 years (OR = 0.132, 95% CI: 0.059 - 0.296, p < 0.001), prophylactic antibiotics use (OR = 0.082, 95% CI: 0.042 - 0.164, p < 0.001) and vacuum sealing drainage use (OR = 0.036, 95% CI: 0.010 - 0.129, p < 0.001) were protective factors. Pathogens results showed that 301 strains of 38 species of bacteria were harvested, among which 178 (59.1%) strains were Gram-positive bacteria, and 123 (40.9%) strains were Gram-negative bacteria. Staphylococcus aureus (108, 60.7%) and Enterobacter cloacae (38, 30.9%) accounted for the largest proportion. The susceptibility of Gram-positive bacteria to Vancomycin and Linezolid was almost 100%. The susceptibility of Gram-negative bacteria to Imipenem, Amikacin, and Meropenem exceeded 73%. CONCLUSION: Orthopedic surgeons need to develop appropriate surgical plans based on the risk factors and protective factors associated with postoperative SSI to reduce its occurrence. Meanwhile, it is recommended to strengthen blood glucose control in the early stage of admission and for surgeons to be cautious and scientific when choosing antibiotic therapy in clinical practice.

Effectiveness and safety of augmentative plating technique in managing nonunion following intramedullary nailing of long bones in the lower extremity: A systematic review and meta-analysis.

PURPOSE: To methodically assess the effectiveness of augmentative plating (AP) and exchange nailing (EN) in managing nonunion following intramedullary nailing for long bone fractures of the lower extremity. METHODS: PubMed, EMBASE, Web of Science, and the Cochrane Library were searched to gather clinical studies regarding the use of AP and EN techniques in the treatment of nonunion following intramedullary nailing of lower extremity long bones. The search was conducted up until May 2023. The original studies underwent an independent assessment of their quality, a process conducted utilizing the Newcastle-Ottawa scale. Data were retrieved from these studies, and meta-analysis was executed utilizing Review Manager 5.3. RESULTS: This meta-analysis included 8 studies involving 661 participants, with 305 in the AP group and 356 in the EN group. The results of the meta-analysis demonstrated that the AP group exhibited a higher rate of union (odds ratio: 8.61, 95% confidence intervals (CI): 4.12 - 17.99, p < 0.001), shorter union time (standardized mean difference (SMD): -1.08, 95 % CI: -1.79 - -0.37, p = 0.003), reduced duration of the surgical procedure (SMD: -0.56, 95 % CI: -0.93 - -0.19, p = 0.003), less bleeding (SMD: -1.5, 95 % CI: -2.81 - -0.18), p = 0.03), and a lower incidence of complications (relative risk: -0.17, 95 % CI: -0.27 - -0.06, p = 0.001). In the subgroup analysis, the time for union in the AP group in nonisthmal and isthmal nonunion of lower extremity long bones was shorter compared to the EN group (nonisthmal SMD: -1.94, 95 % CI: -3.28 - -0.61, p < 0.001; isthmal SMD: -1.08, 95 % CI: -1.64 - -0.52, p = 0.002). CONCLUSION: In the treatment of nonunion in diaphyseal fractures of the long bones in the lower extremity, the AP approach is superior to EN, both intraoperatively (with reduced duration of the surgical procedure and diminished blood loss) and postoperatively (with an elevated union rate, shorter union time, and lower incidence of complications). Specifically, in the management of nonunion of lower extremity long bones with non-isthmal and isthmal intramedullary nails, AP demonstrated shorter union time in comparison to EN.

Transcriptome analysis reveals a lncRNA-miRNA-mRNA regulatory network in OsRpp30-mediated disease resistance in rice.

BACKGROUND: Long non-coding RNAs (lncRNAs) play critical roles in various biological processes in plants. Extensive studies utilizing high-throughput RNA sequencing have revealed that many lncRNAs are involved in plant disease resistance. Oryza sativa RNase P protein 30 (OsRpp30) has been identified as a positive regulator of rice immunity against fungal and bacterial pathogens. Nevertheless, the specific functions of lncRNAs in relation to OsRpp30-mediated disease resistance in rice remain elusive. RESULTS: We conducted a comprehensive analysis of lncRNAs, miRNAs, and mRNAs expression patterns in wild type (WT), OsRpp30 overexpression (OsRpp30-OE), and OsRpp30 knockout (OsRpp30-KO) rice plants. In total, we identified 91 differentially expressed lncRNAs (DElncRNAs), 1671 differentially expressed mRNAs (DEmRNAs), and 41 differentially expressed miRNAs (DEmiRNAs) across the different rice lines. To gain further insights, we investigated the interaction between DElncRNAs and DEmRNAs, leading to the discovery of 10 trans- and 27 cis-targeting pairs specific to the OsRpp30-OE and OsRpp30-KO samples. In addition, we constructed a competing endogenous RNA (ceRNA) network comprising differentially expressed lncRNAs, miRNAs, and mRNAs to elucidate their intricate interplay in rice disease resistance. The ceRNA network analysis uncovered a set of gene targets regulated by lncRNAs and miRNAs, which were found to be involved in pathogen recognition, hormone pathways, transcription factor activation, and other biological processes related to plant immunity. CONCLUSIONS: Our study provides a comprehensive expression profiling of lncRNAs, miRNAs, and mRNAs in a collection of defense mutants in rice. To decipher the putative functional significance of lncRNAs, we constructed trans- and cis-targeting networks involving differentially expressed lncRNAs and mRNAs, as well as a ceRNA network incorporating differentially expressed lncRNAs, miRNAs, and mRNAs. Together, the findings from this study provide compelling evidence supporting the pivotal roles of lncRNAs in OsRpp30-mediated disease resistance in rice.

Identification of two novel rice S genes through combination of association and transcription analyses with gene-editing technology.

Traditional rice blast resistance breeding largely depends on utilizing typical resistance (R) genes. However, the lack of durable R genes has prompted rice breeders to find new resistance resources. Susceptibility (S) genes are potential new targets for resistance genetic engineering using genome-editing technologies, but identifying them is still challenging. Here, through the integration of genome-wide association study (GWAS) and transcriptional analysis, we identified two genes, RNG1 and RNG3, whose polymorphisms in 3'-untranslated regions (3'-UTR) affected their expression variations. These polymorphisms could serve as molecular markers to identify rice blast-resistant accessions. Editing the 3'-UTRs using CRISPR/Cas9 technology affected the expression levels of two genes, which were positively associated with rice blast susceptibility. Knocking out either RNG1 or RNG3 in rice enhanced the rice blast and bacterial blight resistance, without impacting critical agronomic traits. RNG1 and RNG3 have two major genotypes in diverse rice germplasms. The frequency of the resistance genotype of these two genes significantly increased from landrace rice to modern cultivars. The obvious selective sweep flanking RNG3 suggested it has been artificially selected in modern rice breeding. These results provide new targets for S gene identification and open avenues for developing novel rice blast-resistant materials.

[Risk factors for massive hemorrhage after radical nephrectomy and removal of venous tumor thrombus].

OBJECTIVE: To investigate and analyze the risk factors of massive hemorrhage in patients with renal cell carcinoma and venous tumor thrombus undergoing radical nephrectomy and removal of venous tumor thrombus. METHODS: From January 2014 to June 2020, 241 patients with renal cancer and tumor thrombus in a single center of urology at Peking University Third Hospital were retrospectively analyzed. All patients underwent radical nephrectomy and removal of venous tumor thrombus. The relevant preoperative indicators, intraoperative conditions, and postoperative data were statistically analyzed by using statistical software of SPSS 18.0. The main end point of the study was intraoperative bleeding volume greater than 2 000 mL. Logistic regression analysis was used to determine the relevant influencing factors. First, single factor Logistic regression was used for preliminary screening of influencing factors, and variables with single factor Logistic regression analysis P < 0.05 were included in multivariate Logistic regression. In all statistical analyses, P < 0.05 is considered statistically significant. RESULTS: Among the 241 patients included, there were 60 cases of massive hemorrhage, 48 males and 12 females, with a median age of 62 years. The number of non-massive hemorrhage was 181. There were 136 males and 45 females, with a median age of 59 years. Univariate analysis showed that the clinical symptoms (both systemic and local symptoms, OR 2.794, 95%CI 1.087-7.181, P=0.033), surgical approach (open surgery, OR 9.365, 95%CI 4.447-19.72, P < 0.001), Mayo grade (Mayo 3-4, OR 5.257, 95%CI 2.806-10.886, P < 0.001), American Society of Anesthesiologists (ASA) score (ASA level 3, OR 2.842, 95%CI 1.338-6.036, P=0.007), preoperative hemoglobin (OR 0.978, 95%CI 0.965-0.991, P=0.001), preoperative platelet count (OR 0.996, 95%CI 0.992-1.000, P=0.037), maximum tumor thrombus width (OR 1.061, 95%CI 1.033-1.091, P < 0.001), Complicated with bland thrombus (OR 4.493, 95%CI 2.264-8.915, P < 0.001), adrenalectomy (OR 3.101, 95%CI 1.614-5.958, P=0.001), segmental resection of the inferior vena cava (OR 2.857, 95%CI 1.395-5.852, P=0.004). There was a statistically significant difference in these aspects(P < 0.05). Multivariate Logistic regression analysis showed that there was a statistically significant difference in surgical approach (open surgery, OR 6.730, 95%CI 2.947-15.368;P < 0.001), Mayo grade (Mayo 3-4, OR 2.294, 95%CI 1.064-4.948, P=0.034), Complicated with bland thrombus (OR 3.236, 95%CI 1.492-7.020, P=0.003). CONCLUSION: Combining the results of univariate and multivariate Logistic regression analysis, the surgical approach, Mayo grade, and tumor thrombus combined with conventional thrombus were associated risk factors for massive hemorrhage during surgery for renal cell carcinoma with tumor thrombus. Patients who undergo open surgery, high Mayo grade, and tumor thrombus combined with conventional thrombus are at a relatively higher risk of massive hemorrhage.

Expression profiling and function analysis identified new microRNAs regulating cumulus expansion and apoptosis in cumulus cells.

Although microRNAs (miRNAs) expressed in cumulus cells (CCs) may be used to select competent oocytes/embryos, only a limited number of such miRNAs has been reported. To identify more miRNAs that regulate cumulus expansion (CE) and CC apoptosis, we first established that mouse cumulus-oocyte complexes (COCs) cultured in expansion-supporting medium supported full CE while undergoing mild apoptosis, whereas mouse oocytectomized COCs (OOXs) cultured in apoptosis-triggering medium underwent severe apoptosis while supporting no CE. RNA- and miRNA-sequencing and bioinformatics using CCs from these cultured COCs/OOXs identified candidate apoptosis- and/or CE-regulating miRNAs. Transfection of COCs/OOXs with miRNA mimic or inhibitor validated that miR-212-5p and 149-5p promoted CE by facilitating Has2 expression; miR-31-5p and 27a-3p promoted CE by increasing both Has2 and Ptx3 expression; and miR-351-5p and 503-5p inhibited CE by suppressing Ptx3 expression. Furthermore, miR-212-5p, 149-5p and Nov798 inhibited CC apoptosis, involving both Bcl2/Bax and Fas signaling. Analysis using in vivo matured COCs further verified the above apoptosis- and/or CE-regulating miRNAs, except for miR-149-5p. In conclusion, this study identified and validated new CE- and apoptosis-regulating miRNAs in CCs, which could be used as biomarkers to select competent oocytes/embryos and for elucidating how the oocyte-derived factors regulate CE and CC apoptosis.

Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae.

Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.

The WRKY10-VQ8 module safely and effectively regulates rice thermotolerance.

WRKY transcription factors (TFs) play crucial roles in biotic and abiotic stress responses. However, their roles in thermal response are still largely elusive, especially in rice. In this study, we revealed the functions of WRKY10 TF and VQ8 protein containing VQ motif in rice thermotolerance. Overexpression of WRKY10 or loss of VQ8 function increases thermosensitivity, whereas conversely, overexpression of VQ8 or loss of WRKY10 function enhances thermotolerance. Overexpression of WRKY10 accelerates reactive oxygen species (ROS) accumulation in chloroplasts and apoplasts, and it also induces the expression of heat shock TF and protein genes. We also found that WRKY10 regulates nuclear DNA fragmentation and hypersensitive response by modulating NAC4 TF expression. The balance between destructive and protective responses in WRKY10-overexpression plant is more fragile and more easily broken by heat stress compared with wild type. In vitro and in vivo assays revealed that VQ8 interacts with WRKY10 and inhibits the transcription activity via repressing its DNA-binding activity. Our study demonstrates that WRKY10 negatively regulates thermotolerance by modulating the ROS balance and the hypersensitive response and that VQ8 functions antagonistically to positively regulate thermotolerance. The functional module of WRKY10-VQ8 provides safe and effective regulatory mechanisms in the heat stress response.

Phosphorylation-guarded light-harvesting complex II contributes to broad-spectrum blast resistance in rice.

Environmental conditions are key factors in the progression of plant disease epidemics. Light affects the outbreak of plant diseases, but the underlying molecular mechanisms are not well understood. Here, we report that the light-harvesting complex II protein, LHCB5, from rice is subject to light-induced phosphorylation during infection by the rice blast fungus Magnaporthe oryzae We demonstrate that single-nucleotide polymorphisms (SNPs) in the LHCB5 promoter control the expression of LHCB5, which in turn correlates with the phosphorylation of LHCB5. LHCB5 phosphorylation enhances broad-spectrum resistance of rice to M. oryzae through the accumulation of reactive oxidative species (ROS) in the chloroplast. We also show that LHCB5 phosphorylation-induced resistance is inheritable. Our results uncover an immunity mechanism mediated by phosphorylation of light-harvesting complex II.

First Report of Phyllosticta capitalensis Causing Black Spot on Radermachera hainanensis Merr. in China.

Radermachera hainanensis Merr. plants are native in south-central and southeast of China. Plants produce large flowers, and are widely cultivated in China as ornamentals. In April 2020, R. hainanensis Merr. plants grown in Cixi Lvpin Garden (30°26'54″N, 121°25'48″E), Zhejiang Province, were found to have many black circular necrotic lesions. In the early infection stage, the lesions appeared in lower leaves as small black circular spots which developed later into large spots (11 to 38 mm diameter) with grey centers and chlorotic edges. Ultimately, the spots spread and merged. Moreover, infected leaves showed premature leaf fall. Disease intensity reached approximately 20% of plants in the affected field (0.5 ha). After effective chemical control, this disease did not spread to other healthy plants in the same garden. To identify the causative pathogen associated with the disease, ten symptomatic leaves were collected from ten different plants. Leaf tissues were cut from the lesion margins and sterilized as follows: surface sterilized with 75% ethanol for 30 seconds and washed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 3-4 minutes, then washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were cut to small pieces (3×3 mm) and transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under 12 h photoperiod. A total of 15 isolates were obtained from the affected leaves, and all the isolates displayed the same colony characteristics. Then, three single-spore isolates were randomly selected (F2, F5 and F8) for further study. The fungal colonies were dark green with a granular surface, and irregular white edges, later turning black. Conidia were one-celled, oval, and narrow at the end with a single apical end, measuring from 7.8 to 11.1 × 4.6 to 5.9 µm (av. 9.5 × 5.2 µm, n=50). These morphological characteristics were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013; Guarnaccia et al. 2017). The identity of three representative isolates were confirmed by a multilocus approach. The DNA of three isolates were extracted and partial sequences of ribosomal internal transcribed spacer (ITS), actin (ACT), and translation elongation factor 1-alpha (TEF1-α) were amplified and sequenced as previously described (White et al. 1990; O'Donnell et al. 1998; Carbone & Kohn et al. 1999). The three selected isolates shared 100% identical sequence of ITS, ACT and TEF1-α. Then representative isolate F8 was selected for further study. BLAST analysis in GenBank showed that the obtained sequence of ITS (MZ317550) had 99% identity to P. elongata isolate eSX25240811. Other two sequences of ACT (MZ326837) and TEF1-α(MZ326839) showed 99% and 98% identity to P. capitalensis isolate YLWB01, respectively. The phylogenetic trees were constructed by Bootstrap method with 1000 replications using Maximum Likelihood model implemented in the MEGA 7. Results showed that the isolate F8 clustered with P. capitalensis with 100% bootstrap support. Pathogenicity of strain F8 was tested by Koch's postulates. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 28°C. 20 healthy plants were sprayed with a 1×106 conidia ml-1 suspension (three leaves from each individual plants) and another 20 healthy plants were sprayed with sterile distilled water (three leaves from each individual plant) as control. Conidia was obtained from PDA plates after 7 days of incubation in the biochemical incubator at 28°C and concentration was counted in hemacytometer. After 15 days, disease symptoms were observed on all inoculated leaves, whereas the control plants remained asymptomatic. After that, P. capitalensis was re-isolated only from the infected leaves and identified by morphological and sequence analyses. Early identification of P. capitalensis as a causal agent for black spot is crucial to employ effective disease management strategies to control disease in the field. P. capitalensis has been reported on many crops in China (Cheng et al. 2019; Tang et al. 2020; Liao et al. 2020). However, to our knowledge, this is the first report of black spot disease caused by P. capitalensis on Radermachera hainanensis Merr. in China.

In planta transcriptome analysis reveals tissue-specific expression of pathogenicity genes and microRNAs during rice-Magnaporthe interactions.

Transcriptional re-programming in host and pathogen upon leaf and neck infection is an evolving area of research for the rice blast community. Analysis of in planta rice transcriptome in leaf and neck tissues revealed tissue-specific and infection-specific expression of rice and Magnaporthe oryzae genes in host and pathogen. The glycosyl hydrolase, isocitrate lyase, cupin domain containing protein, TF2, CMPG1, CHIT17 and OsCML14 genes were uniquely expressed in leaf infection. Genes like cytochrome P450, inhibitor I family protein, GSTU6, abscisic stress ripening, and cupin domain containing protein were up-regulated during neck infection. In our microRNA sequencing study, Osa-miR166n-3p was highly expressed in upon Magnaporthe leaf infection, whereas osa-miR1661-3p, osa-miR166n-3p and osa-miR159b were overexpressed in neck infection. Here we report several transcripts being targeted by up and down regulated microRNAs during infection. The putative genes expressed upon infection in leaf and neck could be used in understanding the dual-epidemics of blast disease.

Comparative genome analyses of four rice-infecting Rhizoctonia solani isolates reveal extensive enrichment of homogalacturonan modification genes.

BACKGROUND: Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus. RESULTS: Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes. CONCLUSION: Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

The rice RNase P protein subunit Rpp30 confers broad-spectrum resistance to fungal and bacterial pathogens.

RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars.