Elevating plant immunity by translational regulation of a rice WRKY transcription factor.

Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquitin-proteasome system but was accumulated following elicitor or pathogen treatment, whereas the alternate product initiated from the downstream in-frame start codon was stable. Both the full and alternate OsWRKY7 proteins have transcriptional activities in yeast and rice cells, and overexpression of each form enhanced resistance to Xoo infection. Furthermore, disruption of the main AUG in rice increased the endogenous translation of the alternate stabilized form of OsWRKY7 and enhanced bacterial blight resistance. This study provides insights into the coordination of alternative translation and protein stability in the regulation of plant growth and basal defence mediated by the OsWRKY7 transcription factor, and also suggests a promising strategy to breed disease-resistant rice by translation initiation control.

Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection.

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mottle virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.

Ferredoxin 1 is downregulated by the accumulation of abscisic acid in an ABI5-dependent manner to facilitate rice stripe virus infection in Nicotiana benthamiana and rice.

Ferredoxin 1 (FD1) accepts and distributes electrons in the electron transfer chain of plants. Its expression is universally downregulated by viruses and its roles in plant immunity have been brought into focus over the past decade. However, the mechanism by which viruses regulate FD1 remains to be defined. In a previous report, we found that the expression of Nicotiana benthamiana FD1 (NbFD1) was downregulated following infection with potato virus X (PVX) and that NbFD1 regulates callose deposition at plasmodesmata to play a role in defense against PVX infection. We now report that NbFD1 is downregulated by rice stripe virus (RSV) infection and that silencing of NbFD1 also facilitates RSV infection, while viral infection was inhibited in a transgenic line overexpressing NbFD1, indicating that NbFD1 also functions in defense against RSV infection. Next, a RSV-derived small interfering RNA was identified that contributes to the downregulation of FD1 transcripts. Further analysis showed that the abscisic acid (ABA) which accumulates in RSV-infected plants also represses NbFD1 transcription. It does this by stimulating expression of ABA insensitive 5 (ABI5), which binds the ABA response element motifs in the NbFD1 promoter, resulting in negative regulation. Regulation of FD1 by ABA was also confirmed in RSV-infected plants of the natural host rice. The results therefore suggest a mechanism by which virus regulates chloroplast-related genes to suppress their defense roles.

A single amino acid in coat protein of Pepper mild mottle virus determines its subcellular localization and the chlorosis symptom on leaves of pepper.

Pepper mild mottle virus (PMMoV) causes serious economic losses in pepper production in China. In a survey for viral diseases on pepper, two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) were identified with different symptoms in Zhejiang province. Sequence alignment analysis suggested there were only four amino acid differences between the isolates: Val262Gly, Ile629Met and Ala1164Thr in the replicase, and Asp20Asn in the coat protein. Infectious cDNA clones of both isolates were constructed and shown to cause distinctive symptoms. Chlorosis symptoms appeared only on PMMoV-ZJ2-infected plants and the Asp20Asn substitution in the CP was shown to be responsible. Confocal assays revealed that the subcellular localization pattern of the two CPs was different, CP20Asp was mainly located at the cell periphery, whereas most CP20Asn located in the chloroplast. Thus, a single amino acid in the CP determined the chlorosis symptom, accompanied by an altered subcellular localization.

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics.

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC⁻MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV⁻host interaction that might contribute to the symptom development.

Structural characterization of Hericium erinaceus polysaccharides and the mechanism of anti-T2DM by modulating the gut microbiota and metabolites.

A low molecular weight polysaccharides of HEP-1, with molecular weights of 1.67 × 104 Da and composition of →6)-ß-D-Glcp-(1→, →3)-ß-D-Glcp-(1→, ß-D-Glcp-(1→ and →3,6)-ß-D-Glcp-(1→, was isolated and characterized from the fruiting body of Hericium erinaceus. The results indicated that HEP-1 showed potential effects against T2DM-induced imbalance of glucose and lipid metabolism by promoting the serum glucose uptake by hepatic glycogen synthesis via activating the IRS/PI3K/AKT signaling pathway, and inhibiting fatty acid synthesis and reducing hepatic lipid accumulation via activating the AMPK/SREBP-1c signaling pathways. Besides, HEP-1 promoted the production of beneficial bacteria in the gut, and increased the beneficial metabolites in liver through the gut-liver axis, consequently, resisting the occurrence of T2DM.

Stropharia rugoso-annulata acetylated polysaccharides alleviate NAFLD via Nrf2/JNK1/AMPK signaling pathways.

The acetylated Stropharia rugoso-annulata polysaccharides (ASRP) was successfully characterized, and the effects and mechanism on alleviating NAFLD were investigated in HFD-induced mice models. The characterization showed that ASRP was successfully acetylated and rich in galactose. The animal studies demonstrated that ASRP at the dose of 400 mg/kg possessed hepatoprotective effects by potential antioxidation, anti-inflammation and improving hepatocellular histopathology, with the possible mechanisms on regulating the JNK1/AP-1 and activating the Nrf2 signaling pathways. Besides, ASRP could improve the fat metabolism by activating the AMPK/SREBP-1c signaling pathways. The results provided basal theories for the development of ASRP on treating the NAFLD and its complications.

Glucopyranose from Pleurotus geesteranus prevent alcoholic liver diseases by regulating Nrf2/HO-1-TLR4/NF-κB signalling pathways and gut microbiota.

This study investigated the effects of PGPs (Pleurotus geesteranus polysaccharides), a glucopyranose isolated from the mycelium of Pleurotus geesteranus and characterized with the main chain of →4)-α-D-Glcp-(1→, on the prevention against alcohol liver diseases (ALD), with the aim of providing a theoretical basis for the application of P. geesteranus as prebiotic agents in preventing and treating gut dysbiosis and alcohol-related metabolic disorders in individuals with ALD. The results showed that PGP treatment reduced oxidative stress by up-regulating the Nrf2/HO-1 signalling pathways, and decreased the pro-inflammatory factors by down-regulating TLR4/NF-κB signalling pathways. Furthermore, we validated effects of PGPs on balancing the gut-liver axis by maintaining the integrity of the intestinal epithelial barrier of decreasing intestinal permeability, increasing intestinal tight-junction protein and mucin expression and elevating the abundance of short-chain fatty acids (SCFAs) producers in the intestine by regulating the microbiota composition.

Antioxidant and anti-ageing effects of enzymatic polysaccharide from Pleurotus eryngii residue.

The wide industrial cultivation of Pleurotus eryngii (P. eryngii) has resulted in the massive production of mushroom residues (MR) with low-efficiency utilization. In the present study, the P. eryngii enzymatic residue polysaccharide (PERP) was obtained from the P. eryngii residues. The characterization analysis showed that PERP was polysaccharides comprised of five kinds of monosaccharides with molecular weight of 2.05 × 103 Da. PERP also showed rough surface and appeared as spherical structure dispersed in aqueous solution. The animal experiment analysis demonstrated that PERP exhibited potential anti-ageing effects on the brain, liver, kidney and skin, possibly by scavenging reactive radicals, improving the antioxidant status, supressing lipid peroxidation, enhancing organ functions and ameliorating histopathological damage. These results may provide a reference for the efficient utilization of P. eryngii residues in exploring MR-derived functional foods or drugs that delay the ageing process.

Structural characterization and amelioration of sulfated polysaccharides from Ganoderma applanatum residue against CCl4-induced hepatotoxicity.

Natural polysaccharides and their derivatives have attracted academic attention due to their extensive physiological activities. However, the hepatoprotective effects against carbon tetrachloride (CCl4) toxicity have not been well elucidated. The objectives of this study were to characterize the structural properties of sulfated Ganoderma applanatum residue polysaccharides (SGRP) and to evaluate their inhibitory effects on liver fibrosis caused by oxidative stress and inflammation. Our in vivo study showed that SGRP was hepatoprotective in CCl4-induced chronic liver injury mice. It reduced the histopathological damages, down-regulated CYP2E1 (cytochrome P450 2E1) expression, reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, improved the anti-oxidative and anti-inflammatory properties, inhibited TLR4/NF-κB signaling pathway, and reduced the release of inflammatory cytokines. The structural studies indicated that SGRP is a heteropolysaccharide with 7.8% sulfur content and α-linked residue. Our study projects SGRP as a potential candidate in anti-fibrosis treatment by using it as a food supplement or in medicines produced by pharmaceutical industries.

A polysaccharide of PFP-1 from Pleurotus geesteranus attenuates alcoholic liver diseases via Nrf2 and NF-κB signaling pathways.

A polysaccharide named PFP-1 was isolated from the Pleurotus geesteranus fruiting body, and potential investigations on ameliorating oxidative stress and liver injury against alcoholic liver disease (ALD) were performed in mice. The animal studies demonstrated that PFP-1 had hepatoprotective effects by improving hepatocellular histopathology, modulating alcohol metabolisms and restoring the serum lipid levels. Besides, PFP-1 could attenuate oxidative stress and inflammatory responses by activating the Nrf2-mediated signal pathways and regulating the TLR4-mediated NF-κB signal pathways. The characterization indicated that PFP-1 was a typical pyranose-polysaccharide in a triple-helical conformation, which was linked by t-ß-Glcp, 1,6-α-Glcp and 1,2,6-α-Galp. And the characteristic properties of higher water solubility and appropriate molecular weights contributed to the superior bioactivities. The results demonstrated that PFP-1 could be used as a supplement for alleviating alcohol-induced liver damage.

Renoprotective effects of enzyme-hydrolyzed polysaccharides from Auricularia polytricha on adenine-induced chronic kidney diseases in mice.

The present work was aimed to investigate the protective effects of enzymatic-hydrolyzed Auricularia polytricha polysaccharides (EnAPS) on renal functions. The characterizations were analyzed by physicochemical methods, and the renoprotections were processed in adenine-induced chronic kidney diseases (CKD) models of mice. Animal experiments exhibited that EnAPS showed superior renal-protections contributing to its antioxidant effects of increasing the enzyme activities and decreasing the lipid contents, and anti-inflammatory effects of reducing proinflammatory cytokines than A. polytricha polysaccharides (APS). Besides, the anti-apoptosis effects of EnAPS was proved by down-regulating Bax and Caspase-3 expressions and up-regulating Bcl-2 expressions by molecular biotechnology, and the anti-fibrosis effects was confirmed by histopathological observations of staining. The characterizations indicated that lower molecular weights possibly contributed to the superior renoprotective effects. These results suggested that enzymatic hydrolysis had potential effects in enhancing the bioactivities, and the polysaccharides could be used in the development of functional foods supplement against CKD.

Downregulation of Light-Harvesting Complex II Induces ROS-Mediated Defense Against Turnip Mosaic Virus Infection in Nicotiana benthamiana.

The light-harvesting chlorophyll a/b complex protein 3 (LHCB3) of photosystem II plays important roles distributing the excitation energy and modulating the rate of state transition and stomatal response to abscisic acid. However, the functions of LHCB3 in plant immunity have not been well investigated. Here, we show that the expression of LHCB3 in Nicotiana benthamiana (NbLHCB3) was down-regulated by turnip mosaic virus (TuMV) infection. When NbLHCB3 was silenced by tobacco rattle virus-induced gene silencing, systemic infection of TuMV was inhibited. H2O2 was over-accumulated in NbLHCB3-silenced plants. Chemical treatment to inhibit or eliminate reactive oxygen species (ROS) impaired the resistance of the NbLHCB3-silenced plants to TuMV infection. Co-silencing of NbLHCB3 with genes involved in ROS production compromised the resistance of plants to TuMV but co-silencing of NbLHCB3 with genes in the ROS scavenging pathway increased resistance to the virus. Transgenic plants overexpressing NbLHCB3 were more susceptible to TuMV. These results indicate that downregulation of NbLHCB3 is involved in defense against TuMV by inducing ROS production.

Rice stripe virus coat protein induces the accumulation of jasmonic acid, activating plant defence against the virus while also attracting its vector to feed.

The jasmonic acid (JA) pathway plays crucial roles in plant defence against pathogens and herbivores. Rice stripe virus (RSV) is the type member of the genus Tenuivirus. It is transmitted by the small brown planthopper (SBPH) and causes damaging epidemics in East Asia. The role(s) that JA may play in the tripartite interaction against RSV, its host, and vector are poorly understood. Here, we found that the JA pathway was induced by RSV infection and played a defence role against RSV. The coat protein (CP) was the major viral component responsible for inducing the JA pathway. Methyl jasmonate treatment attracted SBPHs to feed on rice plants while a JA-deficient mutant was less attractive than wild-type rice. SBPHs showed an obvious preference for feeding on transgenic rice lines expressing RSV CP. Our results demonstrate that CP is an inducer of the JA pathway that activates plant defence against RSV while also attracting SBPHs to feed and benefitting viral transmission. This is the first report of the function of JA in the tripartite interaction between RSV, its host, and its vector.

Domestic water buffaloes: Access to surface water, disease prevalence and associated economic losses.

Given the shortage and non-availability of freshwater in Pakistan, wastewater is being used for bathing water buffaloes; however, this has a negative impact on animal welfare. Although there is a vast literature on indirect linkages between wastewater and animal productivity, studies focusing on the direct impacts of water buffaloes bathing in wastewater on animal productivity and economic losses are rare. Therefore, using 360 domestic water buffalo farms, this study examines the expenditure and production losses associated with bathing (in wastewater and freshwater) and non-bathing water buffaloes by employing partial budgeting and resource adjustment component techniques. Furthermore, it investigates the prevalence of animal diseases and associated economic effects using correlation analysis and propensity score matching techniques, respectively. The findings reveal that compared to their counterparts (freshwater bathing and non-bathing water buffaloes), buffaloes bathing in wastewater are at increased risk of clinical mastitis, foot and mouth disease (FMD) and tick infestation. Moreover, the use of wastewater for bathing buffaloes also leads to higher economic and production losses by affecting milk productivity, causing premature culling, and reducing slaughter value. The findings of the double-log model show that economic losses are higher if buffaloes bathe in wastewater within 30 min after milking, as there are more chances that those buffaloes would be exposed to bacterial penetration in the teat ducts, which may result in intramammary infection. According to the propensity score matching method, the higher economic damages per month are associated with buffaloes bathing in wastewater and freshwater, 155 and 110 USD per farm, respectively. The study findings reference the need for policies to restrict wastewater access by water buffaloes, and a regular check of and access to cool clean water wallows for bathing during hot summer days, to reduce excess heat and economic losses, and thus improve animal welfare.

Suppression of nbe-miR166h-p5 attenuates leaf yellowing symptoms of potato virus X on Nicotiana benthamiana and reduces virus accumulation.

MicroRNAs (miRNAs) play essential roles in plant development. There is increasing evidence that changed expression of miRNAs in virus-infected plants contributes to the development of viral symptoms. Here, we analysed the altered expression of miRNAs of Nicotiana benthamiana in response to Potato virus X (PVX) by Illumina Solexa sequencing. One of the 21 miRNAs significantly affected, nbe-miR166h-p5, was closely associated with viral symptoms. Using the Tobacco rattle virus-based miRNA suppression (VbMS) system, we found that the suppression of nbe-miR166h-p5 in plants caused leaves to turn dark green with increased chlorophyll. When PVX was inoculated on nbe-miR166h-p5-suppressed plants, the leaf yellowing symptom of PVX was largely attenuated with less reduction in chlorophyll content, and the accumulation of PVX was decreased. nbe-miR166h-p5 was also up-regulated in plants infected by Turnip mosaic virus (TuMV), and its suppression attenuated the leaf yellowing symptom of TuMV and decreased viral accumulation. Three potential targets of nbe-miR166h-p5 were identified. The results indicate the association of nbe-miR166h-p5 with symptoms of PVX and also with those of TuMV, providing useful information on the relationship between miRNA and viral infection.