Transcriptome analysis of genes involved in the pathogenesis mechanism of potato virus Y in potato cultivar YouJin.

Introduction: Potatoes (Solanum tuberosum L.) can be infected by various viruses, but out of all of viruses, the potato virus Y (PVY) is the most detrimental. Research shows that the potato cultivar YouJin is especially vulnerable to PVY and displays severe symptoms, including leaf vein chlorosis, curled leaf margins, large necrotic spots on the leaf blades, and the growth of small new leaves. Methods: PVY infection in potato cultivar YouJin was confirmed through symptom observation, RT-PCR, and Western blot analysis. Transcriptome sequencing was used to analyze the genes associated with PVY pathogenesis in this cultivar. Result: Transcriptome analysis of differential genes was conducted in this study to examine the pathogenesis of PVY on YouJin. The results showed that 1,949 genes were differentially regulated, including 853 upregulated genes and 1,096 downregulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that carbohydrate synthesis and metabolism pathways were suppressed, and electron transferase and hydrolase activities were reduced. Moreover, there were increased expression levels of protein kinase genes. By focusing on plant-pathogen interaction pathways, six core genes all upregulating the WARK family of transcription factors were obtained. Additionally, a constructed PPI network revealed the identification of key modular differential genes, such as downregulated photosynthesis-related protein genes and upregulated AP2/ERF-ERF transcription factors. Functional network enrichment analysis revealed that PVY infection limited RNA metabolism, glutathionylation, and peroxiredoxin activity while triggering the expression of associated defense genes in YouJin. After analyzing the above, 26 DEGs were screened and 12 DEGs were confirmed via RT-qPCR. Conclusion: These results establish a hypothetical framework for clarifying the pathogenesis of PVY in the YouJin variety of potatoes, which will help design the disease resistance of YouJin.

Case report: Lateral medullary syndrome with eight-and-a-half syndromes.

RATIONALE: Lateral medullary syndrome is caused by atherosclerosis or embolism of the vertebral artery and its branches or the posterior inferior cerebellar artery (PICA).The eight-and-a-half syndrome is a rare pontocerebellar nerve-ocular syndrome presenting as a one-and-a-half syndrome plus ipsilateral seventh cerebral nerve palsy. The dorsolateral medullary syndrome combined with the eight-and-a-half syndromes is even rarer, so it is important to recognize the features of the classical brainstem syndrome and the eight-and-a-half syndromes. PATIENT CONCERNS: Most patients with dorsolateral medullary syndrome combined with eight-and-a-half syndromes have a good prognosis, with recovery occurring within a few weeks to a few months, although a few patients may take longer to recover. DIAGNOSIS INTERVENTIONS: In the course of disease development, the patient developed dysarthria, dysphagia, hypothermia, ipsilateral Horner sign and ataxia. Computed tomography was performed which showed cerebral infarction in the left brainstem. Cranial diffusion-weighted imaging + magnetic resonance angiography showed acute infarction in the left cerebellar hemisphere, with a high probability of severe stenosis or occlusion in the intracranial and proximal segments of the basilar arteries. This supports the diagnosis of dorsolateral medullary syndrome. The patient's limited adduction and abduction of the left eye and limited adduction of the right eye, combined with peripheral paralysis of the affected lateral nerve, supported the diagnosis of eight-and-a-half syndromes. The administration of antiplatelet and anti-ester fixation treatment can effectively improve the symptoms and shorten the course of the disease. OUTCOMES: After antiplatelet and anti-ester fixation treatment, the symptoms improved and the patient was discharged. LESSONS: Dorsolateral medullary syndrome combined with eight-and-a-half syndromes is a rare clinical condition, and therefore more attention should be paid to the early diagnosis and treatment of such patients.

Soil microbial diversity under different types of interference in birch secondary forest in the Greater Khingan Mountains in China.

Introduction: Soil microorganisms are an important component of soil ecosystems with an indispensable role in forest ecosystems. We analyzed the soil microbial diversity in birch secondary forest formed by natural restoration or artificial reconstruction after interference by burning, clear cutting, and gradient cutting, and the Betula platyphylla Suk undisturbed forest in the Greater Khingan Mountains in China. Methods: Illumina high-throughput sequencing technology was used to analyze the characteristics of the soil microbial community during the restoration process of birch secondary forest caused by the different types of interference. The relationships between bacteria and fungi were analyzed. The gene functions of the soil bacterial community and the ecological functions of soil fungi were predicted using PICRUSt and FunGuild, respectively. Results: At the phylum level, the species and quantity of bacteria were more abundant than that of fungi. At the genus level, no obvious differences in the abundance of bacteria were observed; there were obvious differences in the abundance of fungi. Among the eight sample plots, the artificial larch forest belt had the highest bacterial and fungal alpha diversity, which was slightly higher than undisturbed forest, while the other sample plots were significantly lower. Gradual cutting pure birch forest bacteria and fungi had the highest beta diversity, and artificial larch forest belt bacteria and heavy burn sample plot fungi had the lowest beta diversity. Samples from the cutting and burning sample plots were significantly different from the undisturbed forest at the phylum level of Acidobacteriae, Acidimicrobiia, Mortierellomycetes and Sordariomycetes. We found statistical differences in biomarkers between bacterial and fungal communities in undisturbed forest and artificial larch forest belt and burn sample plots. PICRUSt prediction and FunGuild prediction showed that soil bacterial and fungal communities were rich in gene and ecological functions, respectively. In the microbial network, the stability or anti-interference performance of the fungal community was higher than that of bacteria. Conclusion: Our data reveal the characteristics of the soil microbial community during the restoration process of Betula platyphylla Suk secondary forest under different types of disturbance, which is of great significance for understanding the role of soil microorganisms in the forest ecological cycle.

A patient with MELAS syndrome combined with autoimmune abnormalities: a case report.

Background: Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) is a group of maternally inherited disorders caused by mutations or deletions in mitochondrial genes with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes as the main clinical manifestations. Case presentation: We reported a 20-year-old female patient with MELAS syndrome combined with autoimmune abnormalities. She suffered from an intermittent headache in the right temporal region with no obvious cause, and then, after strenuous exercise in dance class, the headache became aggravated, accompanied by unresponsiveness, blurred vision, and diplopia. Her blood lactate levels were elevated, her antinuclear antibodies were positive, and the antimetabolic glutamate receptors 5 in her serum were positive. Brain DWI showed a hypertensive signal in the right temporo-parietal-occipital cortex and subcortical area. Brain MRS showed decreased NAA peak and increased Lac peak. Muscle biopsy showed myogenic damage, and the modified Gomori trichrome (MGT) staining showed ragged red fibers (RRF). A genetic study revealed a mitochondrial DNA A3243G mutation. Conclusion: Mitochondrial encephalomyopathy is a rare clinical condition; however, the association with autoimmune diseases is not yet clear and still needs further research and analysis.

Rhamnolipid-induced alleviation of bioclogging in Managed Aquifer Recharge (MAR): Interactions with bacteria and porous media.

The prevention and treatment of bioclogging is of great significance to the application of Managed Aquifer Recharge (MAR). This study investigated the alleviating effect of biosurfactant rhamnolipid (RL) on bioclogging by laboratory-scale percolation experiments. The results show that the addition of RL greatly reduced bioclogging. Compared with the group without RL, the relative hydraulic conductivity (K') of the 100 mg/L RL group increased 5 times at the end of the experiment (23 h), while the bacterial cell amount and extracellular polymeric substances (EPS) content on the sand column surface (0-2 cm) decreased by 60.8% and 85.7%, respectively. In addition, the richness and diversity of the microbial communities within the clogging matter decreased after the addition of RL. A variety of bacterial phyla were found, among which Proteobacteria were predominant in all groups. At the genus level, RL reduced the relative abundance of Acinetobacter, Bacillus, Klebsiella, and Pseudomonas. These microbes are known as strong adhesion, large size, and easy to form biofilms, therefore playing a critical role during MAR bioclogging. Moreover, RL changed the surface properties of bacteria and porous media, which results in the increase of electrostatic repulsion and decrease of hydrophobic interaction between them. Therefore, RL mediated the bacteria-porous media interaction to reduce biomass in porous media, thereby alleviating bioclogging. This study implies that RL's addition is an environmentally friendly and effective method to alleviate the bioclogging in MAR.

Carrier burden of over 300 diseases in Han Chinese identified by expanded carrier testing of 300 couples using assisted reproductive technology.

BACKGROUND: Expanded carrier screening (ECS) has become a common practice for identifying carriers of monogenic diseases. However, existing large gene panels are not well-tailored to Chinese populations. In this study, ECS testing for pathogenic variants of both single-nucleotide variants (SNVs) and copy number variants (CNVs) in 330 genes implicated in 342 autosomal recessive (AR) or X-linked diseases was carried out. We assessed the differences in allele frequencies specific to the Chinese population who have used assisted reproductive technology (ART) and the important genes to screen for in this population. METHODOLOGY: A total of 300 heterosexual couples were screened by our ECS panel using next-generation sequencing. A customed bioinformatic algorithm was used to analyze SNVs and CNVs. Guidelines from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology were adapted for variant interpretation. Pathogenic or likely pathogenic (P/LP) SNVs located in high homology regions/deletions and duplications of one or more exons in length were independently verified with other methods. RESULTS: 64.83% of the patients were identified to be carriers of at least one of 342 hereditary conditions. We identified 622 P/LP variants, 4.18% of which were flagged as CNVs. The rate of at-risk couples was 3%. A total of 149 AR diseases accounted for 64.05% of the cumulative carrier rate, and 48 diseases had a carrier rate above 1/200 in the test. CONCLUSION: An expanded screening of inherited diseases by incorporating different variant types, especially CNVs, has the potential to reduce the occurrence of severe monogenic diseases in the offspring of patients using ART in China.

RpoZ regulates 2,4-DAPG production and quorum sensing system in Pseudomonas fluorescens 2P24.

Introduction: Pseudomonas fluorescens 2P24 was isolated from soil of natural decay associated with wheat take-all and it can effectively control soil-borne diseases caused by a variety of plant pathogens. 2,4-diacetylphloroglucinol (2,4-DAPG), is produced by P. fluorescens 2P24 and plays an important role in the prevention and control of plant diseases. To understand the resistant mechanism, in this study, we conducted experiments to explore the regulation role of rpoZ in the synthesis of the antibiotic 2,4-DAPG and regulation of QS system. Methods: A random mini-Tn5 mutagenesis procedure was used to screen regulators for phlA transcription in stain PM901, which containing a phlA∷lacZ transcriptional fusion reporter plasmid. We identified 12 insertion mutants could significantly change phlA gene expression. By analyzing the amino acid sequences of the interrupted gene, we obtained a mutant strain Aa4-29 destroyed the rpoZ gene, which encodes the omiga subunit. We constructed the plasmid of rpoZ mutant (pBBR-△rpoZ) transformed into competent cells of P. fluorescens 2P24 by electro-transformation assay. The strains of P. fluorescens 2P24/pBBR, 2P24-△rpoZ/pBBR, 2P24-△rpoZ/pBBR-rpoZ were used to evaluate the regulation role of rpoZ in 2,4-DAPG production and quorum sensing system. Results: According to ß-galactosidase activity, we found that rpoZ positively regulated the expression of phlA (a synthesis gene of 2,4-DAPG) and PcoI (a synthesis gene of PcoI/PcoR QS signal system) at the transcriptional level. The production of 2,4-DAPG antibiotic and signal molecule AHL was influenced by rpoZ. Further, rpoZ was involved in regulating rsmA expression. RpoZ also has a certain regulatory effect on rpoS transcription, but no effect on the transcription of phlF, emhABC and emhR. According to the biocontrol assay, P. fluorescens 2P24 strains with rpoZ showed obvious antagonism ability against the Rhizoctonia solani in cotton, while the mutant strain of rpoZ lost the biocontrol effect. RpoZ had a significant effect on the swimming and biofilm formation in P. fluorescens 2P24. Conclusion: Our data showed that rpoZ was an important regulator of QS system, 2,4-DAPG in P. fluorescens 2P24. This may imply that P. fluorescens 2P24 has evolved different regulatory features to adapt to different environmental threats.

Strategies for Engineering Virus Resistance in Potato.

Potato (Solanum tuberosum L.) is an important vegetable crop that plays a pivotal role in the world, especially given its potential to feed the world population and to act as the major staple food in many developing countries. Every year, significant crop loss is caused by viral diseases due to a lack of effective agrochemical treatments, since only transmission by insect vectors can be combated with the use of insecticides, and this has been an important factor hindering potato production. With the rapid development of molecular biology and plant genetic engineering technology, transgenic approaches and non-transgenic techniques (RNA interference and CRISPR-cas9) have been effectively employed to improve potato protection against devastating viruses. Moreover, the availability of viral sequences, potato genome sequences, and host immune mechanisms has remarkably facilitated potato genetic engineering. In this study, we summarize the progress of antiviral strategies applied in potato through engineering either virus-derived or plant-derived genes. These recent molecular insights into engineering approaches provide the necessary framework to develop viral resistance in potato in order to provide durable and broad-spectrum protection against important viral diseases of solanaceous crops.

Methyltransferase-like (METTL) homologues participate in Nicotiana benthamiana antiviral responses.

Methyltransferase (MTase) enzymes catalyze the addition of a methyl group to a variety of biological substrates. MTase-like (METTL) proteins are Class I MTases whose enzymatic activities contribute to the epigenetic and epitranscriptomic regulation of multiple cellular processes. N6-adenosine methylation (m6A) is a common chemical modification of eukaryotic and viral RNA whose abundance is jointly regulated by MTases and METTLs, demethylases, and m6A binding proteins. m6A affects various cellular processes including RNA degradation, post-transcriptional processing, and antiviral immunity. Here, we used Nicotiana benthamiana and plum pox virus (PPV), an RNA virus of the Potyviridae family, to investigated the roles of MTases in plant-virus interaction. RNA sequencing analysis identified MTase transcripts that are differentially expressed during PPV infection; among these, accumulation of a METTL gene was significantly downregulated. Two N. benthamiana METTL transcripts (NbMETTL1 and NbMETTL2) were cloned and further characterized. Sequence and structural analyses of the two encoded proteins identified a conserved S-adenosyl methionine (SAM) binding domain, showing they are SAM-dependent MTases phylogenetically related to human METTL16 and Arabidopsis thaliana FIONA1. Overexpression of NbMETTL1 and NbMETTL2 caused a decrease of PPV accumulation. In sum, our results indicate that METTL homologues participate in plant antiviral responses.

Whole-genome analysis revealed the growth-promoting and biological control mechanism of the endophytic bacterial strain Bacillus halotolerans Q2H2, with strong antagonistic activity in potato plants.

Introduction: Endophytes are colonizers of healthy plants and they normally exhibit biocontrol activities, such as reducing the occurrence of plant diseases and promoting plant growth. The endophytic bacterium Bacillus halotolerans Q2H2 (Q2H2) was isolated from the roots of potato plants and was found to have an antagonistic effect on pathogenic fungi. Methods: Q2H2 was identified by morphological observations, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Genes related to the anti-fungal and growth-promoting effects were analyzed using whole-genome sequencing and comparative genomic analysis. Finally, we analyzed the growth-promoting and biocontrol activities of Q2H2 in potato plants using pot experiments. Results: Antagonism and non-volatile substance plate tests showed that Q2H2 had strong antagonism against Fusarium oxysporum, Fusarium commune, Fusarium graminearum, Fusarium brachygibbosum, Rhizoctonia solani and Stemphylium solani. The plate test showed that Q2H2 had the ability to produce proteases, cellulases, ß-1,3-glucanase, dissolved organic phosphate, siderophores, indole-3-acetic acid (IAA), ammonia and fix nitrogen. The suitable growth ranges of Q2H2 under different forms of abiotic stress were pH 5-9, a temperature of 15-30°C, and a salt concentration of 1-5%. Though whole-genome sequencing, we obtained sequencing data of approximately 4.16 MB encompassed 4,102 coding sequences. We predicted 10 secondary metabolite gene clusters related to antagonism and growth promotion, including five known products surfactin, bacillaene, fengycin, bacilysin, bacillibactin, and subtilosin A. Average nucleotide identity and comparative genomic analyses revealed that Q2H2 was Bacillus halotolerans. Through gene function annotation, we analyzed genes related to antagonism and plant growth promotion in the Q2H2 genome. These included genes involved in phosphate metabolism (pstB, pstA, pstC, and pstS), nitrogen fixation (nifS, nifU, salA, and sufU), ammonia production (gudB, rocG, nasD, and nasE), siderophore production (fhuC, fhuG, fhuB, and fhuD), IAA production (trpABFCDE), biofilm formation (tasA, bslA, and bslB), and volatile compound production (alsD, ilvABCDEHKY, metH, and ispE), and genes encoding hydrolases (eglS, amyE, gmuD, ganB, sleL, and ydhD). The potato pot test showed that Q2H2 had an obvious growth-promoting effect on potato roots and better control of Fusarium wilt than carbendazim. Conclusion: These findings suggest that the strain-specific genes identified in bacterial endophytes may reveal important antagonistic and plant growth-promoting mechanisms.

A case of EBV encephalomyelitis with positive anti-GFAP-IgG antibody with recurrent fever and dysuresia as the main symptoms: Case report and retrospective analysis.

RATIONALE: Due to neuronal destruction caused by Epstein-Barr virus (EBV) infection, exposure to neuronal surface antigens may lead to an imbalance in immune tolerance, which in turn triggers an autoimmune response. In addition, due to the involvement of nonspecific B-cell activation or molecular mimicry, EBV and Glial Fibrillary Acidic Protein (GFAP) receptors may have common epitopes. Viral infection triggers activation of B-cell and cross-reaction with viral antibodies, resulting in autoimmune encephalomyelitis. The clinical presentation may be complex or even atypical. A small number of patients may develop autoimmune reactions. PATIENT CONCERNS: Most patients with EBV encephalomyelitis have a good prognosis, with the disease generally having a short course, few complications, and a good prognosis. In most patients, after treatment, their neurological function basically recovers within a few weeks or months. DIAGNOSIS INTERVENTIONS: The patient had fever and headache. His 3 tests for cerebral spinal fluid (CSF) are consistent with the features of viral encephalomyelitis. Pathogenic examination of CSF confirmed EBV, and imaging suggested brain and spinal cord involvement. After antiviral treatment, the patient's symptoms relieved. The diagnosis of EBV encephalomyelitis was considered. However, the patient's temperature continued to increase. He was transferred to a superior hospital and was given GFAP-Ab in CSF, which was strongly positive. The patient was given immunoglobulin and antiviral therapy. This supports the diagnosis of GFAP-IgG antibody positive with EBV encephalomyelitis. OUTCOMES: After treatment with antiviral drugs and immunoglobulins, the patient's symptoms improved and he was able to function. LESSONS: EBV encephalomyelitis is a rare clinical disease. Therefore, more attention should be paid to the early diagnosis and treatment of similar patients to avoid misdiagnosis. CSF tests, genetic tests, and imaging tests can confirm the diagnosis.

Whole-genome analysis revealed the growth-promoting mechanism of endophytic bacterial strain Q2H1 in potato plants.

Introduction: Endophytes are non-pathogenic inhabitants of healthy plant tissues and have been found to promote plant growth and health. The endophytic bacterial strain Q2H1 was isolated from the roots of the potato and was identified to exhibit growth-promoting effects in potato plants. Methods: Whole-genome sequencing was performed to reveal the mechanism underlying its growth-promoting effect. The obtained sequencing data of approximately 5.65 MB encompassed 5,533 coding sequences. Of note, nine secondary metabolite gene clusters, including siderophore gene clusters, closely associated with plant growth promotion (PGP) were predicted by antiSMASH software. Comparative genomic analysis revealed that Q2H1 belongs to the genus Peribacillus. By gene function annotation, those genes related to plant growth-promoting activities, including indole-3-acetic acid (IAA) synthesis in tryptophan metabolism, siderophore biosynthetic activity, phosphate solubilization, nitrogen fixation, and related genes, were summarized. IAA (14.4 µg/ml) was presumptively produced by Q2H1 using the Salkowski colorimetric method. A total of five genes, namely, phoU, pstB, pstA1, pstC, and pstS, were annotated for phosphate solubilization, which is associated with the ability of the Q2H1 strain to solubilize phosphate under in vitro conditions. Results: It is revealed that genes in the Q2H1 genome associated with nitrogen fixation belonged to three groups, namely, nitrogen fixation (nifU, sufU, salA, and nifS), nitrogen metabolism (nirA, nrtB, and nasA), and glutamate synthesis (glnA, gltB, gltD, and gudB), supported by evidence that Q2H1 grew on medium without nitrogen. We have also identified a siderophore gene cluster located on the chromosome of Q2H1, including seven genes (viz., rbsR, rhbf, rhbE, rhbD, rhbC, rhbA, ddc, and an unknown gene). In the in vitro assay, a prominent brown circle around the colony was produced on the chrome azurol S medium at 48 and 72 h post-inoculation, indicating that the siderophore gene cluster in Q2H1 harbored the ability to produce siderophores. Conclusion: In summary, these findings implied that identifying strain-specific genes for their metabolic pathways in bacterial endophytes may reveal a variety of significant functions of plant growth-promoting mechanisms.

Case report: A late-onset cobalamin C defect first presenting as a depression in a teenager.

Background: The cobalamin C (cblC) defect, a common inborn disorder of cobalamin metabolism due to a genetic mutation in MMACHC, can cause combined methylmalonic acid and homocysteine accumulation in blood, urine, or both. In this article, a late-onset case was reported, and the patient first presented with depression identified with the MMACHC gene. We summarized the clinical features of the cblC defect, the relationship between genotype and phenotype, and the clinical experience concerning the diagnosis and treatment of the cblC defect. Case presentation: Initially presented with depression, the 16-year-old female patient showed progressive abnormal gait and bilateral lower limb weakness after 3 months. Blood routine examination suggested severe hyperhomocysteinemia, and screening for urine organic acids found elevated methylmalonic acid. Family gene sequencing showed mutations detected in MMACHC. She had a compound heterozygous mutation, while the c.271dupA (p.R91Kfs∗14) was only detected in her father and the c.482 G>A (p.R161Q) was only detected in her mother. Hence, she was diagnosed with a cblC defect and treated with B vitamin supplements. The muscle strength of both lower limbs improved notably. Conclusion: This case indicated that depression could be a presenting sign of cblC-type methylmalonic aciduria and homocysteinemia, and enhanced the genotype-phenotype relationship of the cblC defect, which will contribute to further understanding of this emerging disease.

AntiGQ1b antibody positive with MFS/GBS overlapped syndrome with diplopia and hemiplegia onset: Case report and retrospective analysis.

RATIONALE: GBS and MFS have been divided into several subtypes, constituting a series of independent and overlapping syndromes that share similar pathophysiology, leading to common clinical features, including history of previous infection, single-phase course, symmetry, skull or limbs weakness, CFS albumin cell separation (high protein, normal cell count), antiganglioside antibodies and axon, or evidence of demyelinating neuropathy neurophysiology. Part of the MFS in patients with clinical manifestations may be complicated, and even symptoms are not typical. A few patients may overlap with BBE or GBS. PATIENT CONCERNS: Most patients with MFS/GBS overlap syndrome have a good prognosis, and a few patients may experience fluctuations or re-exacerbations. In most patients, after treatment, their neurological function basically recovers within a few weeks or months. DIAGNOSIS INTERVENTIONS: The patient had ophthalmoplegia, ataxia, weak force, and protein-cell separation in cerebrospinal fluid during the development of the disease. The diagnosis of MFS overlapped with typical GBS was considered. The CSF specific IgG oligoclonal zone and anti-Sulfatide antibody were positive. Anti-GT1a IgG was positive. Anti-GQ1b IgG was positive, which supported the diagnosis of GBS spectrum disorders. According to their common immunological basis, plasma exchange or intravenous immunoglobulin (IVIG) therapy is recommended, which can effectively improve the symptoms and shorten the course of the disease. OUTCOMES: After treatment with glucocorticoids and gamma globulin, the symptoms improved and the patient was discharged. LESSONS: MFS/GBS Superimposed syndrome is a rare clinical disease. Therefore, more attention should be paid to early diagnosis and treatment of similar patients to avoid misdiagnosis. Cerebral spinal fluid (CFS) examination, neuroelectrophysiology, and GQ1b antibody detection can be used to confirm the diagnosis.

Targeting of genomic and negative-sense strands of viral RNA contributes to antiviral resistance mediated by artificial miRNAs and promotes the emergence of complex viral populations.

Technology based on artificial small RNAs, including artificial microRNAs (amiRNAs), exploits natural RNA silencing mechanisms to achieve silencing of endogenous genes or pathogens. This technology has been successfully employed to generate resistance against different eukaryotic viruses. However, information about viral RNA molecules effectively targeted by these small RNAs is rather conflicting, and factors contributing to the selection of virus mutants escaping the antiviral activity of virus-specific small RNAs have not been studied in detail. In this work, we transformed Nicotiana benthamiana plants with amiRNA constructs designed against the potyvirus plum pox virus (PPV), a positive-sense RNA virus, and obtained lines highly resistant to PPV infection and others showing partial resistance. These lines have allowed us to verify that amiRNA directed against genomic RNA is more efficient than amiRNA targeting its complementary strand. However, we also provide evidence that the negative-sense RNA strand is cleaved by the amiRNA-guided RNA silencing machinery. Our results show that the selection pressure posed by the amiRNA action on both viral RNA strands causes an evolutionary explosion that results in the emergence of a broad range of virus variants, which can further expand in the presence, and even in the absence, of antiviral challenges.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N6 -methyladenosine (m6 A). m6 A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus-the largest genus of plant RNA viruses-and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus. We show that Nicotiana benthamiana m6 A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus-infected samples showed that m6 A peaks are enriched in plant transcript 3' untranslated regions and in discrete internal and 3' terminal regions of PPV and PVY genomes. Down-regulation of N. benthamiana AlkB homologues of the plant-specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6 A implication and the proviral roles of AlkB homologues in Potyvirus infection.

The Reversible Methylation of m6A Is Involved in Plant Virus Infection.

In recent years, m6A RNA methylation has attracted broad interest and is becoming a hot research topic. It has been demonstrated that there is a strong association between m6A and viral infection in the human system. The life cycles of plant RNA viruses are often coordinated with the mechanisms of their RNA modification. Here, we reviewed recent advances in m6A methylation in plant viruses. It appears that m6A methylation plays a dual role during viral infection in plants. On the one hand, m6A methylation acts as an antiviral immune response induced by virus infection, which inhibits viral replication or translation through the methylation of viral genome RNAs. On the other hand, plant viruses could disrupt the m6A methylation through interacting with the key proteins of the m6A pathway to avoid modification. Those plant viruses containing ALKB domain are discussed as well. Based on this mechanism, we propose that new strategies for plant virus control could be designed with competitive antagonists of m6A-associated proteins.

Inhibitory Effect of Osthole from Cnidium monnieri (L.) Cusson on Fusarium oxysporum, a Common Fungal Pathogen of Potato.

Fusarium wilt of potato is one of the most common diseases of potato in China, and is becoming a serious threat in potato production. It has been reported that osthole from Cnidium monnieri (L.) Cusson can inhibit plant pathogens. Here, we test the anti-fungal activity of C. monnieri osthole against Fusarium oxysporum in potatoes. The results showed that at a concentration of 5 mg/mL, osthole was able to obviously inhibit mycelial growth of F. oxysporum. We found that osthole caused changes of mycelial morphology, notably hyphal swelling and darkening. Osthole significantly reduced the spore germination of Fusarium by 57.40%. In addition, osthole also inhibited the growth of other pathogens such as Fusarium moniliforme J. Sheld, Thanatephorus cucumeris Donk, and Alternaria alternata (Fr.) Keissl, but not Alternaria solani Jonesetgrout and Valsa mali Miyabe and G. Yamada. Our results suggest that osthole has considerable potential as an agent for the prevention and treatment of potato Fusarium wilt.

Molecular and biological characterization of Potato virus Y detected in zucchini in China.

Potato virus Y (PVY) is an important viral pathogen of the plant family Solanaceae. However, it has not been reported that PVY infects zucchini (Cucurbita pepo L). Zucchini plants showing symptoms of leaf mosaic and fruit ringspot were collected from Inner Mongolia, China. Using RT-PCR, we amplified the PVY-specific bands in zucchini (PVY-Zu isolate). Further, we found that the viral pathogen could infect Nicotiana benthamiana via mechanical inoculation with the extract of the zucchini leaves. Filamentous virions were observed in infected N. benthamiana plants under a transmission electron microscope. Additionally, the complete genomic fragment of the PVY-Zu isolate was obtained via RT-PCR and RACE analysis, containing 9662 nucleotides (nt) excluding the 3'-terminal poly (A) tail. The 5' and 3' untranslated regions (UTRs) were 150 nt and 329 nt, respectively, while the putative polyprotein contained 3061 amino acids. Comparison of the PVY-Zu isolate with 19 other PVY isolates revealed that they shared nucleotide sequence identities of ~ 83.1-99.8% at the complete genomic level. Based on the complete genome sequences, phylogenetic analysis showed that PVY-Zu clustered with PVY strain N605 (X97895-PVY), a Swiss necrotic isolate of PVY. Compared with the complete genome sequence of X97895-PVY, the PVY-Zu isolate had a deletion of a 39-nt fragment in its 5' UTR and 22 mutated nucleotides without changes in the amino acids. Moreover, although the similarity in the identities of the PVY-Zu isolate and PVY N isolate N-Jg (AY166867) was about 96.2%, the former had a deletion of a 38-nt fragment in its 5' UTR and 334 mutations among the viral genome, which resulted in 61 amino acid changes. However, we could not detect any recombination throughout the PVY-Zu genome. To our knowledge, this is the first study reporting that PVY could infect zucchini plants, thus broadening the host diversity of the viral pathogen. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-020-00647-2.

Virus Host Jumping Can Be Boosted by Adaptation to a Bridge Plant Species.

Understanding biological mechanisms that regulate emergence of viral diseases, in particular those events engaging cross-species pathogens spillover, is becoming increasingly important in virology. Species barrier jumping has been extensively studied in animal viruses, and the critical role of a suitable intermediate host in animal viruses-generated human pandemics is highly topical. However, studies on host jumping involving plant viruses have been focused on shifting intra-species, leaving aside the putative role of "bridge hosts" in facilitating interspecies crossing. Here, we take advantage of several VPg mutants, derived from a chimeric construct of the potyvirus Plum pox virus (PPV), analyzing its differential behaviour in three herbaceous species. Our results showed that two VPg mutations in a Nicotiana clevelandii-adapted virus, emerged during adaptation to the bridge-host Arabidopsis thaliana, drastically prompted partial adaptation to Chenopodium foetidum. Although both changes are expected to facilitate productive interactions with eIF(iso)4E, polymorphims detected in PPV VPg and the three eIF(iso)4E studied, extrapolated to a recent VPg:eIF4E structural model, suggested that two adaptation ways can be operating. Remarkably, we found that VPg mutations driving host-range expansion in two non-related species, not only are not associated with cost trade-off constraints in the original host, but also improve fitness on it.