Identification of Crucial Genes and Regulatory Pathways in Alfalfa against Fusarium Root Rot.

Fusarium root rot, caused by Fusarium spp. in alfalfa (Medicago sativa L.), adversely impacts alfalfa by diminishing plant quality and yield, resulting in substantial losses within the industry. The most effective strategy for controlling alfalfa Fusarium root rot is planting disease-resistant varieties. Therefore, gaining a comprehensive understanding of the mechanisms underlying alfalfa's resistance to Fusarium root rot is imperative. In this study, we observed the infection process on alfalfa seedling roots infected by Fusarium acuminatum strain HM29-05, which is labeled with green fluorescent protein (GFP). Two alfalfa varieties, namely, the resistant 'Kangsai' and the susceptible 'Zhongmu No. 1', were examined to assess various physiological and biochemical activities at 0, 2, and 3 days post inoculation (dpi). Transcriptome sequencing of the inoculated resistant and susceptible alfalfa varieties were conducted, and the potential functions and signaling pathways of differentially expressed genes (DEGs) were analyzed through gene ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Meanwhile, a DEG co-expression network was constructed though the weighted gene correlation network analysis (WGCNA) algorithm. Our results revealed significant alterations in soluble sugar, soluble protein, and malondialdehyde (MDA) contents in both the 'Kangsai' and 'Zhongmu No. 1' varieties following the inoculation of F. acuminatum. WGCNA analysis showed the involvement of various enzyme and transcription factor families related to plant growth and disease resistance, including cytochrome P450, MYB, ERF, NAC, and bZIP. These findings not only provided valuable data for further verification of gene functions but also served as a reference for the deeper explorations between plants and pathogens.

NLRs derepress MED10b- and MED7-mediated repression of jasmonate-dependent transcription to activate immunity.

Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b, which confers resistance to tospovirus, as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus. MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b-MED7-JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus. Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.

First Report of Leaf Spot Disease Caused by Epicoccum nigrum on Smooth Bromegrass (Bromus inermis) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an excellent forage species widely distributed in Gansu, Qinghai, Inner Mongolia, and other provinces of China (Gong et al. 2019). In July 2021, typical leaf spot symptoms were observed on the leaves of smooth bromegrass plants in Ewenki Banner of Hulun Buir, China (49°5'8″N, 119°44'28″E, alt. 622.5 m). Approximately 90% of plants were affected, with symptoms apparent throughout the plant but mainly concentrated on the lower middle leaves. We collected 11 plants to identify the causal pathogen of leaf spot on smooth bromegrass. Samples (5×5 mm) of symptomatic leaves were excised and surface-sanitized with 75% ethanol for 3 min, rinsed three times with sterile distilled water, and incubated on water agar (WA) at 25℃ for three days. The lumps were cut along the edges and transplanted to potato dextrose agar (PDA) for subculture. After two purification cultures, ten strains, termed HE2 to HE11, were collected. The front side of the colony morphology was cottony or woolly, the center was greyish-green, circled with greyish-white color, with reddish pigmentation on the reverse. The conidia were globose or subglobose, yellow-brown or dark brown, with surface verrucae, and 23.89±3.76×20.28±3.23 µm (n = 50) in size. The morphological characteristics of the mycelia and conidia of the strains mtched those of Epicoccum nigrum (El-Sayed et al. 2020). The primers ITS1/ITS4 (White et al. 1991), LROR/LR7 (Rehner and Samuels 1994), 5F2/7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009) were used to amplify and sequence four phylogenic loci (ITS, LSU, RPB2 and ß-tubulin), respectively. The sequences of ten strains have been deposited in GenBank, and the detailed accession numbers were shown in Table S1. BLAST analysis of these sequences showed 99-100%, 96-98%, 97-99% and 99-100% homology with the E. nigrum strain in the ITS, LSU, RPB2 and TUB sequenced regions, respectively. The sequences of ten test strains and other Epicoccum spp. strains obtained from GenBank were aligned by ClustalW by MEGA (version 11.0) software. After a series of alignment, cutting and splicing, the phylogenetic tree was constructed by the neighbor-joining method with 1000 bootstrap replicates based on the ITS, LSU, RPB2, and TUB sequences. The test strains were clustered together with E. nigrum, with branch support rate of 100%. Combined with morphological and molecular biological characteristics, ten strains were identified as E. nigrum. For the pathogenicity test, the seeds of smooth bromegrass were soaked for four days and then sown into six pots (10 cm diameter × 15 cm height) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 60% relative humidity. Microconidia of the strain produced on wheat bran medium after 10 days were washed with sterile deionized water, filtered through three layers of sterile cheese cloth, quantified, and the concentration adjusted to 1 × 106 microconidia/ml with a hemocytometer. When the plants had grown to a height of about 20 cm, the leaves of plants in three pots were sprayed with the spore suspension, 10 mL per pot, while the remaining three pots were inoculated with sterile water and served as controls (LeBoldus and Jared 2010). The inoculated plants were cultured in an artificial climate box under a 16-h photoperiod with temperatures of 24°C and 60% relative humidity. Brown spots were apparent on the leaves of the treated plants after five days, whereas the leaves of the controls remained healthy. The same E. nigum strain were re-isolated from the inoculated plants and identified by the morphological and molecular techniques described above. To our knowledge, this is the first report of leaf spot disease caused by E. nigrum on smooth bromegrass in China, as well as in the world. Infection with this pathogen could reduce the yield and quality of smooth bromegrass production. For this reason, strategies for the management and control of this disease should be developed and implemented.

NLR surveillance of pathogen interference with hormone receptors induces immunity.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.

Deep Learning Algorithm for Online College Physical Education Teaching with Flipping Classroom.

In view of the inability to accurately analyze the application of deep learning in college physical education teaching design from the perspective of flipping classroom, this paper puts forward an improved deep learning method based on the integration of flipping classroom vision and deep learning, which can reduce the design ability of physical education teaching design in college physical education teaching design and improve the level of college physical education teaching design. Firstly, the initial data set is established by using the theory of flipping classroom horizon, so that the data meet the requirements of normal distribution and reduce the differences between teaching data; Then, the physical education teaching design is divided into different subdesigns by using the theory of flipping classroom horizon. Find the best design result in this domain in each subinstructional design; Finally, under the guidance of the theory of flipping classroom horizon, each subdesign realizes the optimal allocation of teaching resources. MATLAB simulation shows that under the conditions of initial design scheme and teaching resources setting, the improved deep learning method can improve the accuracy of physical education teaching design and shorten the convergence time of design, which is superior to the original deep learning method. Therefore, the deep learning method is used to analyze the instructional design of college physical education, which has a good design effect and is suitable for the instructional design of college physical education.

Degradation pathways of penthiopyrad by δ-MnO2 mediated processes: a combined density functional theory and experimental study.

Penthiopyrad is a widely used succinate dehydrogenase inhibitor (SDHI) fungicide and frequently detected in natural environments. In order to better understand its fate in natural systems, the degradation of penthiopyrad by manganese dioxide (MnO2) was investigated in this study. The results show that penthiopyrad is rapidly degraded in the δ-MnO2 system. Moreover, density functional theory (DFT) calculations reveal that the atoms of C18, C12, and S1 in penthiopyrad have relatively high reactive active sites. The degradation products mainly include sulfoxides, sulfones, and diketone. A sulfoxide and sulfone are formed by the oxidation of the thioether group, and diketone is formed by the oxidation of the olefin group, respectively. Based on the DFT calculations and degradation products, the degradation pathway of penthiopyrad by MnO2 is proposed. This study also reveals that the degradation of penthiopyrad by δ-MnO2 is affected by various environmental factors. A warm environment, low pH, and co-existing humic acid are beneficial to the degradation of penthiopyrad in the δ-MnO2 system, whereas, co-existing metal cations inhibit penthiopyrad degradation. This result provides theoretical guidance for predicting the potential fate of penthiopyrad in natural environments.

Characterization of the Roles of SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 in Sw-5b-Mediated Resistance to Tomato Spotted Wilt Virus.

The tomato Sw-5b gene confers resistance to tomato spotted wilt virus (TSWV) and encodes a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal Solanaceae-specific domain (SD). Although our understanding of how Sw-5b recognizes the viral NSm elicitor has increased significantly, the process by which Sw-5b activates downstream defense signaling remains to be elucidated. In this study, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate the roles of the SGT1/RAR1, EDS1/NDR1, NPR1, and NRC/ADR1/NRG1 genes in the Sw-5b-mediated signaling pathway. We found that chaperone SGT1 was required for Sw-5b function, but co-chaperone RAR1 was not. Sw-5b-mediated immune signaling was independent of both EDS1 and NDR1. Silencing NPR1, which is a central component in SA signaling, did not result in TSWV systemic infection in Sw-5b-transgenic N. benthamiana plants. Helper NLR NRCs (NLRs required for cell death) were required for Sw-5b-mediated systemic resistance to TSWV infection. Suppression of NRC2/3/4 compromised the Sw-5b resistance. However, the helper NLRs ADR1 and NRG1 may not participate in the Sw-5b signaling pathway. Silencing ADR1, NRG1, or both genes did not affect Sw-5b-mediated resistance to TSWV. Our findings provide new insight into the requirement for conserved key components in Sw-5b-mediated signaling pathways.

The Sw-5b NLR nucleotide-binding domain plays a role in oligomerization, and its self-association is important for activation of cell death signaling.

Plant and animal intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors play important roles in sensing pathogens and activating defense signaling. However, the molecular mechanisms underlying the activation of host defense signaling by NLR proteins remain largely unknown. Many studies have determined that the coil-coil (CC) or Toll and interleukin-1 receptor/resistance protein (TIR) domain of NLR proteins and their dimerization/oligomerization are critical for activating downstream defense signaling. In this study, we demonstrated that, in tomato, the nucleotide-binding (NB) domain Sw-5b NLR alone can activate downstream defense signaling, leading to elicitor-independent cell death. Sw-5b NB domains can self-associate, and this self-association is crucial for activating cell death signaling. The self-association was strongly compromised after the introduction of a K568R mutation into the P-loop of the NB domain. Consequently, the NBK568R mutant induced cell death very weakly. The NBCΔ20 mutant lacking the C-terminal 20 amino acids can self-associate but cannot activate cell death signaling. The NBCΔ20 mutant also interfered with wild-type NB domain self-association, leading to compromised cell death induction. By contrast, the NBK568R mutant did not interfere with wild-type NB domain self-association and its ability to induce cell death. Structural modeling of Sw-5b suggests that NB domains associate with one another and likely participate in oligomerization. As Sw-5b-triggered cell death is dependent on helper NLR proteins, we propose that the Sw-5b NB domain acts as a nucleation point for the assembly of an oligomeric resistosome, probably by recruiting downstream helper partners, to trigger defense signaling.

The Intracellular Immune Receptor Sw-5b Confers Broad-Spectrum Resistance to Tospoviruses through Recognition of a Conserved 21-Amino Acid Viral Effector Epitope.

Plants use both cell surface-resident pattern recognition receptors (PRRs) and intracellular nucleotide binding leucine-rich repeat (NLR) receptors to detect various pathogens. Plant PRRs typically recognize conserved pathogen-associated molecular patterns (PAMPs) to provide broad-spectrum resistance. By contrast, plant NLRs generally detect pathogen strain-specific effectors and confer race-specific resistance. Here, we demonstrate that the tomato (Solanum lycopersicum) NLR Sw-5b confers broad-spectrum resistance against American-type tospoviruses by recognizing a conserved 21-amino acid peptide region within viral movement protein NSm (NSm21). Sw-5b NB-ARC-LRR domains directly associate with NSm21 in vitro and in planta. Domain swap, site-directed mutagenesis and structure modeling analyses identified four polymorphic sites in the Sw-5b LRR domain that are critical for the recognition of NSm21 Furthermore, recognition of NSm21 by Sw-5b likely disturbs the residues adjacent to R927 in the LRR domain to weaken the intramolecular interaction between LRR and NB-ARC domains, thus translating recognition of NSm21 into activation of Sw-5b. Natural variation analysis of Sw-5b homologs from wild tomato species of South America revealed that the four polymorphic sites in the Sw-5b LRR domain were positively selected during evolution and are all necessary to confer resistance to tospovirus. The results described here provide a new example of a plant NLR mediating broad-spectrum resistance through recognition of a small conserved PAMP-like region within the pathogen effector.

Development of a lateral-flow assay (LFA) for rapid detection of Soybean mosaic virus.

Soybean mosaic virus (SMV) is the most common virus in soybean and poses a serious threat to crop production and germplasm recession in many countries worldwide. In this study, a highly practical and rapid lateral-flow assay (LFA) was developed for the detection of SMV. The SMV coat protein (CP) was prokaryotically expressed and purified to immunize mice. After generation of hybridoma cell lines, four anti-SMV monoclonal antibodies were selected. The LFA-strip was then assembled using a double-antibody sandwich strategy. When the SMV-infected leaf sample was assayed using the assembled LFA-strip, the positive pink color appeared in the test line within 5-10min. The strip only gave positive results with SMV and not other viruses tested and could be used to detect 800 fold dilutions of infected leaf samples. The LFA could be used to detect SMV in infected leaf tissue as well as soybean seeds. To our knowledge, this is the first report of the development of a LFA for the detection of SMV. The practical, rapid and specific assay that was developed in this study can be widely applied to the diagnosis and surveillance of SMV in the laboratory and the field.

Changes of sarcoplamic reticular Ca(2+)-ATPase and IP(3)-I receptor mRNA expression in patients with atrial fibrillation.

OBJECTIVE: To investigate changes in the expression of sarcoplamic reticular Ca(2+)-ATPase (SERCA) and IP(3)-I receptors (IP(3)R(1)) mRNA in patients with atrial fibrillation. METHODS: Thirty-eight patients with mitral stenosis undergoing open heart surgery were studied. 100 mg of atrial tissue was obtained during surgery from the right appendage and the right atrium. The amount of messenger ribonucleic acid (mRNA) amount of SERCA and IP(3)R(1) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RESULTS: Levels of mRNA expression of SERCA in patients with AF, as compared with subjects in sinus rhythm, was lower and that of IP(3)R(1) was higher. The longer AF was sustained, the higher the levels of mRNA. There was no significant difference between right atrial free wall and right appendage. CONCLUSIONS: The expression changes of SERCA and IP3R mRNA may correlate with the initiation or maintenance of AF.