A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

MAPKs trigger antiviral immunity by directly phosphorylating a rhabdovirus nucleoprotein in plants and insect vectors.

Signaling by the evolutionarily conserved mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) plays critical roles in converting extracellular stimuli into immune responses. However, whether MAPK/ERK signaling induces virus immunity by directly phosphorylating viral effectors remains largely unknown. Barley yellow striate mosaic virus (BYSMV) is an economically important plant cytorhabdovirus that is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a propagative manner. Here, we found that the barley (Hordeum vulgare) MAPK MPK3 (HvMPK3) and the planthopper ERK (LsERK) proteins interact with the BYSMV nucleoprotein (N) and directly phosphorylate N protein primarily on serine 290. The overexpression of HvMPK3 inhibited BYSMV infection, whereas barley plants treated with the MAPK pathway inhibitor U0126 displayed greater susceptibility to BYSMV. Moreover, knockdown of LsERK promoted virus infection in SBPHs. A phosphomimetic mutant of the N Ser290 (S290D) completely abolished virus infection because of impaired self-interaction of BYSMV N and formation of unstable N-RNA complexes. Altogether, our results demonstrate that the conserved MAPK and ERK directly phosphorylate the viral nucleoprotein to trigger immunity against cross-kingdom infection of BYSMV in host plants and its insect vectors.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

CCR4, a RNA decay factor, is hijacked by a plant cytorhabdovirus phosphoprotein to facilitate virus replication.

Carbon catabolite repression 4 (CCR4) is a conserved mRNA deadenylase regulating posttranscriptional gene expression. However, regulation of CCR4 in virus infections is less understood. Here, we characterized a pro-viral role of CCR4 in replication of a plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV). The barley (Hordeum vulgare) CCR4 protein (HvCCR4) was identified to interact with the BYSMV phosphoprotein (P). The BYSMV P protein recruited HvCCR4 from processing bodies (PBs) into viroplasm-like bodies. Overexpression of HvCCR4 promoted BYSMV replication in plants. Conversely, knockdown of the small brown planthopper CCR4 inhibited viral accumulation in the insect vector. Biochemistry experiments revealed that HvCCR4 was recruited into N-RNA complexes by the BYSMV P protein and triggered turnover of N-bound cellular mRNAs, thereby releasing RNA-free N protein to bind viral genomic RNA for optimal viral replication. Our results demonstrate that the co-opted CCR4-mediated RNA decay facilitates cytorhabdovirus replication in plants and insects.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43-55) and the remaining region (aa 56-295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

[Study on phosphate removal and recovery by activated alumina].

Activated alumina was studied for removing phosphate from water, and the recovery of adsorbed phosphate on activated aluminum oxide was also tested. Phosphate solution was prepared using distilled water, tap water and Luoshijiang River water, respectively. All the phosphate adsorption tests using activated alumina were proved to be well fitted with Langmuir isotherm and the respective maximum adsorption amount were 20.88, 32.15 and 29.85 mg x g(-1), respectively. The presence of electrolyte in water could be a positive factor for phosphate removal. As the pH value of phosphate solution became lower the Zeta potential of activated alumina increased, which could enhance the phosphate removal efficiency of activated alumina. The recovery tests indicated that NaOH (0.1 mol x L(-1)) solution could almost completely extract the phosphate adsorbed by activated alumina.

[Start-up characteristics of the anaerobic reactor seeded with immobilized microorganisms].

In order to overcome the disadvantages of the anaerobic reactor such as slow growth and long start-up, the flocculent anaerobic sludge was embedded and used as the seed sludge in the anaerobic treatment of PTA wastewater with the objective of keeping biomass in the reactor. The start-up characteristics of the UASB reactor were investigated. During the 136 days' running, COD removal rate of PTA wastewater achieved 75%-85% at the volumetric loading rate (COD) of 3 kg x (m3 x d)(-1) and the hydraulic retention time (HRT) of 3-4 day. The anaerobic system had good stability and biomass retaining ability. On the other hand, variations of EPS, SEM observation and methanogens DNA in sludge granules verified the growth of immobilized bacteria in both quantity and microorganism morphology, although mass transfer through the immobilization media was to some degree limited.

Simultaneous removal of ammonium and phosphate by zeolite synthesized from coal fly ash as influenced by acid treatment.

Zeolite synthesized from fly ash (ZFA) without modification is not efficient for the purification of NH4+ and phosphate at low concentrations that occur in real effluents, despite the high potential removal capacity. To develop an effective technique to enhance the removal efficiency of ammonium and phosphate at low concentrations, ZFA was modified with acid treatment and the simultaneous removal of ammonium and phosphate in a wide range of concentration was investigated. It was seen that when compared with untreated ZFA, only the treatment by 0.01 mol/L of H2SO4 significantly improved the removal efficiency of ammonium at low initial concentrations. The behavior was well explained by the pH effect. Treatment by more concentrated H2SO4 led to the deterioration of the ZFA structure and a decrease in the cation exchange capacity. Treatment by 0.01 mol/L H2SO4 improved the removal efficiency of phosphate by ZFA at all initial P concentrations, while the treatment by concentrated H2SO4 (> or = 0.9 mol/L) resulted in a limited maximum phosphate immobilization capacity (PIC). It was concluded that through a previous mild acid treatment (e.g. 0.01 mol/L of H2SO4), ZFA can be used in the simultaneous removal of NH4+ and P at low concentrations in simulating real effluent.

Production and application of anaerobic granular sludge produced by landfill.

Sludge granulation is considered to be the most critical parameter governing successful operation of an upflow anaerobic sludge blanket and expanded granular sludge bed (EGSB) reactors. Pre-granulated seeding sludge could greatly reduce the required startup time. Two lab-scale and a pilot-scale EGSB reactors were operated to treat Shaoxing Wastewater Treatment Plant (SWWTP) containing wastewater from real engineering printing and dyeing with high pH and sulfate concentration. The microbiological structure and the particle size distribution in aerobic excess sludge, sanitary landfill sludge digested for one year, and the granular sludge of EGSB reactor after 400 d of operation were analyzed through scanning electron microscopy (SEM) and sieves. The lab-scale EGSB reactor seeded with anaerobic sludge after digestion for one year in landfill showed obviously better total chemical oxygen demand (TCOD) removal efficiency than one seeded with aerobic excess sludge after cation polyacrylamide flocculation-concentration and dehydration. The TCOD removed was 470.8 mg/L in pilot scale EGSB reactor at short hydraulic retention time of 15 h. SEM of sludge granules showed that the microbiological structure of the sludge from different sources showed some differences. SEM demonstrated that Methanobacterium sp. was present in the granules of pilot-scale EGSB and the granular sludge produced by landfill contained a mixture of anaerobic/anoxic organisms in abundance. The particle size distribution in EGSB demonstrated that using anaerobic granular sludge produced by sanitary landfill as the seeding granular sludge was feasible.

Removal of organic matter and nitrogen from distillery wastewater by a combination of methane fermentation and denitrification/nitrification processes.

The distillery wastewater of Guangdong Jiujiang Distillery, which is characteristic of containing high organic matters and rich total nitrogen, was treated by a combination of methane fermentation and denitrification/nitrification processes. 80% of COD in the raw wastewater was removed by methane fermentation at the COD volumetric loading rate of 20 kg COD/(m3 x d) using the expanded granule sludge bed (EGSB) process. However, almost all the organic nitrogen in the raw wastewater was converted into ammonia by ammonification there. Ammonia and volatile fatty acids (VFA) remaining in the anaerobically treated wastewater were simultaneously removed utilizing VFA as an electron donor by denitrification occurring in the other EGSB reactor and nitrification using PEG-immobilized nitrifying bacteria with recirculation process. An aerobic biological contact oxidization reactor was designed between denitrification/nitrification reactor for further COD removal. With the above treatment system, 18000-28000 mg/L of COD in raw wastewater was reduced to less than 100 mg/L. Also, ammonia in the effluent of the system was not detected and the system had a high removal rate for 900-1200 mg/L of TN in the raw wastewater, only leaving 400 mg/L of nitrate nitrogen.

[Pilot study on the biochemical treatment of mother liquid of polyvinyl chloride produced by suspension polymerization].

In the pilot study on the treatment of mother liquid of polyvinyl chloride produced by suspension polymerization (SPVC) by using a novel aerobic treatment system-4 cascade aerobic biofilm reactors with internal circulation, the removal efficiencies of the COD and turbidity at different HRT together with the resistance impact of the system were researched. The laws of the biological growth and the development were observed. Some primary factors (suspended solids, chironomidae larvae, nutrition) influencing the steady operation of the reactor were studied and their controlling methods were suggested. The experiment results show that the start-up period was very short and the total COD removal rate was over 75% at HRT of 14 h, the effluent less than 50 mg/L can steadily meet the requirement of wastewater discharge standard and also be reused after advanced treatment.