Transgenic Potato with Resistant to PVX，PVY and PSTVd Simultaneously by amiRNA-mediated and Resistance Evaluation

peer reviewedIn order to obtain potato plants which could be resistant to multiple viruses ( viroid) simultaneous- ly，P25，HC-Pro，and Virp1 gene targeting PVX，PVY，and PSTVd proteins were cloned，respectively. Three types of amiRNAs targeting sequences encoding the silencing suppressor P25，HC-Pro and Virp1 were designed by using Ar- abidopsis thaliana miR159a as backbone. These three amiRNA sequences were connected by Overlapping PCR. The synthetic of P25，HC-Pro and Virp1 gene was inserted into the expression vector pCAMBIA1300-221 to form p1300-221-preamiR-P25-HCPro-Virp1，and the vector was verified by PCR and restriction enzyme digestion. Pre-a- miR-P25-HCPro-Virp1 was transformed into minituber of potato cultivar Favorita by Agrobacterium tumefaciens in- fection. 15 transformed plants were obtained through regenerating，pressure screening and differentiation. PCR re- sults showed that 10 of them were detected the same size fragment ( 729 bp) as the target gene. Further qRT-PCR t e s t i n g c o n f i r m e d t h a t a m i R - P 2 5 - H C P r o - V i r p 1 g e n e w a s e x p r e s s e d i n 1 0 t r a n s f o r m e d p l a n t s ，a n d t h e r e l a t i v e e x p r e s - sion was between 7. 68 - 21. 37. Inoculated the T0 transgenic plants with the mixture virus of PVX，PVY and PSTVd by friction into the leaves of the plants at the 6 - 8 leaf stage. The plant growth was observed after 20 days and the results showed that the control plants were severely susceptible，with symptoms such as short plants and mottled leaves，while the transformed plants did not show infection symptoms，and growth normally. There was no vi- rus detected by RT-PCR either. This indicated that the transfected amiR-P25-HCPro-Virp could be stably expressed in the transformed plants，and the transformed plants were resistant to PVX，PVY and PVSTd viruses ( viroid) . Transformed plants resistant to PVX，PVY and PVSTd were obtained，and the resistance was significant，which pro- vided new genetic resources for potato virus-resistant breeding

CMV2b-AGO Interaction Is Required for the Suppression of RDR-Dependent Antiviral Silencing in Arabidopsis.

peer reviewedUsing a transient plant system, it was previously found that the suppression of Cucumber mosaic virus (CMV) 2b protein relies on its double-strand (ds) RNA binding capacity, but it is independent of its interaction with ARGONAUTE (AGO) proteins. Thus, the biological meaning of the 2b-AGO interaction in the context of virus infection remains elusive. In this study, we created infectious clones of CMV mutants that expressed the 2b functional domains of dsRNA or AGO binding and tested the effect of these CMV mutants on viral pathogenicity. We found that the mutant CMV2b(1-76) expressing the 2b dsRNA-binding domain exhibited the same virulence as wild-type CMV in infection with either wild-type Arabidopsis or rdr1/6 plants with RDR1- and RDR6-deficient mutations. However, remarkably reduced viral RNA levels and increased virus (v)siRNAs were detected in CMV2b(1-76)-infected Arabidopsis in comparison to CMV infection, which demonstrated that the 2b(1-76) deleted AGO-binding domain failed to suppress the RDR1/RDR6-dependent degradation of viral RNAs. The mutant CMV2b(8-111) expressing mutant 2b, in which the N-terminal 7 amino acid (aa) was deleted, exhibited slightly reduced virulence, but not viral RNA levels, in both wild-type and rdr1/6 plants, which indicated that 2b retained the AGO-binding activity acquired the counter-RDRs degradation of viral RNAs. The deletion of the N-terminal 7 aa of 2b affected virulence due to the reduced affinity for long dsRNA. The mutant CMV2b(18-111) expressing mutant 2b lacked the N-terminal 17 aa but retained its AGO-binding activity greatly reduced virulence and viral RNA level. Together with the instability of both 2b(18-111)-EGFP and RFP-AGO4 proteins when co-expressed in Nicotiana benthamiana leaves, our data demonstrates that the effect of 2b-AGO interaction on counter-RDRs antiviral defense required the presence of 2b dsRNA-binding activity. Taken together, our findings demonstrate that the dsRNA-binding activity of the 2b was essential for virulence, whereas the 2b-AGO interaction was necessary for interference with RDR1/6-dependent antiviral silencing in Arabidopsis

Replication of a pathogenic non-coding RNA increases DNA methylation in plants associated with a bromodomain-containing viroid-binding protein.

peer reviewedViroids are plant-pathogenic molecules made up of single-stranded circular non-coding RNAs. How replicating viroids interfere with host silencing remains largely unknown. In this study, we investigated the effects of a nuclear-replicating Potato spindle tuber viroid (PSTVd) on interference with plant RNA silencing. Using transient induction of silencing in GFP transgenic Nicotiana benthamiana plants (line 16c), we found that PSTVd replication accelerated GFP silencing and increased Virp1 mRNA, which encodes bromodomain-containing viroid-binding protein 1 and is required for PSTVd replication. DNA methylation was increased in the GFP transgene promoter of PSTVd-replicating plants, indicating involvement of transcriptional gene silencing. Consistently, accelerated GFP silencing and increased DNA methylation in the of GFP transgene promoter were detected in plants transiently expressing Virp1. Virp1 mRNA was also increased upon PSTVd infection in natural host potato plants. Reduced transcript levels of certain endogenous genes were also consistent with increases in DNA methylation in related gene promoters in PSTVd-infected potato plants. Together, our data demonstrate that PSTVd replication interferes with the nuclear silencing pathway in that host plant, and this is at least partially attributable to Virp1. This study provides new insights into the plant-viroid interaction on viroid pathogenicity by subverting the plant cell silencing machinery

The Identification of Lymphocyte-Like Cells and Lymphoid-Related Genes in Amphioxus Indicates the Twilight for the Emergency of Adaptive Immune System

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergency of AIS before the invertebrate-vertebrate transition during evolution

Occurrence and molecular characterization of Potato spindle tuber viroid (PSTVd) isolates from potato plants in North China

peer reviewedChina is the largest potato producing country worldwide, with this crop representing the fourth largest staple food crop in China. However, the steady presence of Potato spindle tuber viroid (PSTVd) over the past five decades has a significant economic impact on potato production. To determine why PSTVd control measures have been ineffective in China, more than 1 000 seed potatoes collected between 2009 and 2014 were subjected to PSTVd detection at the Supervision and Testing Center for Virus-free Seed Potatoes Quality, Ministry of Agriculture, China. A high PSTVd infection rate (6.5%) was detected among these commercial seed potatoes. Some breeding lines of potato collected from 2012 to 2015 were also tested for PSTVd infection, revealing a high rate of PSTVd contamination in these potato propagation materials. Furthermore, comparison of the full-length sequences of 71 different Chinese PSTVd isolates revealed a total of 74 predominant PSTVd variants, which represented 42 different sequence variants of PSTVd. Comparative sequence analysis revealed 30 novel PSTVd sequence variants specific to China. Comprehensive phylogenetic analysis uncovered a close relationship between the Chinese PSTVd sequence variants and those isolated from Russia. It is worth noting that three intermediate strains and six mild strains were identified among these variants. These results have important implications for explaining the ineffective control of PSTVd in China and thus could serve as a basic reference for designing more effective measures to eliminate PSTVd from China in the future

Arbuscular mycorrhizal fungi increased peanut (Arachis hypogaea L.) yield by changing the rhizosphere microbial community structure in saline-alkali soil

Arbuscular mycorrhizal fungi (AMF) have demonstrated the potential to enhance the saline-alkali tolerance in plants. Nevertheless, the extent to which AMF can ameliorate the tolerance of salt-sensitive plants to alkaline conditions necessitates further investigation. The current study is primarily centered on elucidating the impact of AMF on the growth of the Huayu22 (H22) when cultivated in saline-alkaline soil. We leveraged DNA of rhizosphere microorganisms extracted from saline-alkali soil subjected to AMF treatment and conducted high-throughput sequencing encompassing 16S rRNA gene and ITS sequencing. Our findings from high-throughput sequencing unveiled Proteobacteria and Bacillus as the prevailing phylum and genus within the bacterial population, respectively. Likewise, the predominant fungal phylum and genus were identified as Ascomycota and Haematonectria. It is noteworthy that the relative abundance of Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Ascomycota exhibited significant increments subsequent to AMF inoculation. Our investigation into soil enzyme activity revealed a remarkable surge post-AMF inoculation. Notably, the amounts of pathogen growth inhibitory enzymes and organic carbon degrading enzymes rise, as predicted by the putative roles of microbial communities. In saline-alkali soil, inoculation of AMF did boost the yield of H22. Notable improvements were observed in the weight of both 100 fruits and 100 grains, which increased by 20.02% and 22.30%, respectively. Conclusively, this study not only provides a theoretical framework but also furnishes empirical evidence supporting the utilization of AMF as a viable strategy for augmenting the yield of salt-sensitive plants grown in alkaline conditions

The Reputation Analysis Based on the Signal Game Theory Model of the Aquatic Products of Logistics Company

Part 1: Simulation, Optimization, Monitoring and Control TechnologyInternational audienceThis article examines the reputation of the aquatic product logistics companies based on the signal model. We use the model to study these companies in separation equilibrium and pooling equilibrium situation and provide a static economic analysis. Suggestions are presented in the end to improve the reputation of these companies

Numerical simulation of slab edging process by the Reproducing Kernel Particle Method considering friction effect

A mesh-free approach is used for analysing the effect of the friction factor on three-dimensional steady state slab edging, which is based on the Reproducing Kernel Particle Method (RKPM, Liu et al., 1995) and the material flow formulation for slightly compressible materials (Osakada et al., 1982). In order to cope with the singularity at the corner of the roll entry, a simple technique with a very thin array of cells at the inlet region adjacent to the plastic deformation zone (Xiong et al., 2003) is used. The results show that the dog-bone shape becomes smaller with the increment of friction factor. The roll separating force and total rolling torque increase with the friction factor