A Quantum Mechanics-Based Method to Predict Intramolecular Hydrogen Bond Formation Reflecting P-glycoprotein Recognition.

Passive membrane permeability and an active transport process are key determinants for penetrating the blood-brain barrier. P-glycoprotein (P-gp), a well-known transporter, serves as the primary gatekeeper, having broad substrate specificity. A strategy to increase passive permeability and impair P-gp recognition is intramolecular hydrogen bonding (IMHB). 3 is a potent brain penetrant BACE1 inhibitor with high permeability and low P-gp recognition, although slight modifications to its tail amide group significantly affect P-gp efflux. We hypothesized that the difference in the propensity to form IMHB could impact P-gp recognition. Single-bond rotation at the tail group enables both IMHB forming and unforming conformations. We developed a quantum-mechanics-based method to predict IMHB formation ratios (IMHBRs). In a given data set, IMHBRs accounted for the corresponding temperature coefficients measured in NMR experiments, correlating with P-gp efflux ratios. Furthermore, the method was applied in hNK2 receptor antagonists, demonstrating that the IMHBR could be applied to other drug targets involving IMHB.

Aspartic protease inhibitor enhances resistance to potato virus Y and A in transgenic potato plants.

BACKGROUND: Viruses are the major threat to commercial potato (Solanum tuberosum) production worldwide. Because viral genomes only encode a small number of proteins, all stages of viral infection rely on interactions between viral proteins and host factors. Previously, we presented a list of the most important candidate genes involved in potato plants' defense response to viruses that are significantly activated in resistant cultivars. Isolated from this list, Aspartic Protease Inhibitor 5 (API5) is a critical host regulatory component of plant defense responses against pathogens. The purpose of this study is to determine the role of StAPI5 in defense of potato against potato virus Y and potato virus A, as well as its ability to confer virus resistance in a transgenic susceptible cultivar of potato (Desiree). Potato plants were transformed with Agrobacterium tumefaciens via a construct encoding the potato StAPI5 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. RESULTS: Transgenic plants overexpressing StAPI5 exhibited comparable virus resistance to non-transgenic control plants, indicating that StAPI5 functions in gene regulation during virus resistance. The endogenous StAPI5 and CaMV 35S promoter regions shared nine transcription factor binding sites. Additionally, the net photosynthetic rate, stomatal conductivity, and maximum photochemical efficiency of photosystem II were significantly higher in virus-infected transgenic plants than in wild-type plants. CONCLUSION: Overall, these findings indicate that StAPI5 may be a viable candidate gene for engineering plant disease resistance to viruses that inhibit disease development.

Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral noncovalent, nonpeptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel noncovalent inhibitor could be a potential oral agent for treating COVID-19.

Effect of aphid biology and morphology on plant virus transmission.

Aphids severely affect crop production by transmitting many plant viruses. Viruses are obligate intracellular pathogens that mostly depend on vectors for their transmission and survival. A majority of economically important plant viruses are transmitted by aphids. They transmit viruses either persistently (circulative or non-circulative) or non-persistently. Plant virus transmission by insects is a process that has evolved over time and is strongly influenced by insect morphological features and biology. Over the past century, a large body of research has provided detailed knowledge of the molecular processes underlying virus-vector interactions. In this review, we discuss how aphid biology and morphology can affect plant virus transmission. © 2021 Society of Chemical Industry.

Resistance induction based on the understanding of molecular interactions between plant viruses and host plants.

BACKGROUND: Viral diseases cause significant damage to crop yield and quality. While fungi- and bacteria-induced diseases can be controlled by pesticides, no effective approaches are available to control viruses with chemicals as they use the cellular functions of their host for their infection cycle. The conventional method of viral disease control is to use the inherent resistance of plants through breeding. However, the genetic sources of viral resistance are often limited. Recently, genome editing technology enabled the publication of multiple attempts to artificially induce new resistance types by manipulating host factors necessary for viral infection. MAIN BODY: In this review, we first outline the two major (R gene-mediated and RNA silencing) viral resistance mechanisms in plants. We also explain the phenomenon of mutations of host factors to function as recessive resistance genes, taking the eIF4E genes as examples. We then focus on a new type of virus resistance that has been repeatedly reported recently due to the widespread use of genome editing technology in plants, facilitating the specific knockdown of host factors. Here, we show that (1) an in-frame mutation of host factors necessary to confer viral resistance, sometimes resulting in resistance to different viruses and that (2) certain host factors exhibit antiviral resistance and viral-supporting (proviral) properties. CONCLUSION: A detailed understanding of the host factor functions would enable the development of strategies for the induction of a new type of viral resistance, taking into account the provision of a broad resistance spectrum and the suppression of the appearance of resistance-breaking strains.

Balancing potency and basicity by incorporating fluoropyridine moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitor that affords robust and sustained central Aß reduction.

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aß reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.

Identification of a defense response gene involved in signaling pathways against PVA and PVY in potato.

Potato is the most important non-grain food crop in the world. Viruses, particularly potato virus Y (PVY) and potato virus A (PVA), are among the major agricultural pathogens causing severe reduction in potato yield and quality worldwide. Virus infection induces host factors to interfere with its infection cycle. Evaluation of these factors facilitates the development of intrinsic resistance to plant viruses. In this study, a small G-protein as one of the critical signaling factors was evaluated in plant response to PVY and PVA to enhance resistance. For this purpose, the gene expression dataset of G-proteins in potato plant under five biotic (viruses, bacteria, fungi, nematodes, and insects) and four abiotic (cold, heat, salinity, and drought) stress conditions were collected from gene expression databases. We reduced the number of the selected G-proteins to a single protein, StSAR1A, which is possibly involved in virus inhibition. StSAR1A overexpressed transgenic plants were created via the Agrobacterium-mediated method. Real-time PCR and Enzyme-linked immunosorbent assay tests of transgenic plants mechanically inoculated with PVY and PVA indicated that the overexpression of StSAR1A gene enhanced resistance to both viruses. The virus-infected transgenic plants exhibited a greater stem length, a larger leaf size, a higher fresh/dry weight, and a greater node number than those of the wild-type plants. The maximal photochemical efficiency of photosystem II, stomatal conductivity, and net photosynthetic rate in the virus-infected transgenic plants were also obviously higher than those of the control. The present study may help to understand aspects of resistance against viruses.

Prevalência de hiperglicemia de estresse em uma unidade de terapia intensiva / Prevalence of stress hyperglycemia in an intensive care unit

Objetivo: Avaliar a prevalência e o manejo da hiperglicemia de estresse em pacientes internados em uma unidade de terapia intensiva. Métodos: Estudo retrospectivo, realizado de janeiro a junho de 2018. Os dados foram obtidos a partir de 582 prontuá- rios eletrônicos, considerando os valores glicêmicos durante a hospitalização, história prévia ou não de diabetes mellitus, causas do internamento, tempo de permanência na unidade de terapia intensiva, presença de complicações durante o internamento e conduta utilizada em caso de hiperglicemia de estresse. Resulta- dos: Dos 582 pacientes internados na unidade de terapia intensi- va, 579 tiveram sua glicemia indicada nos prontuários analisados; 341 (58,9%) apresentaram hiperglicemia em algum momento da internação, sendo a hiperglicemia de estresse caracterizada em 200 pacientes (35%). A duração média de internamento desses pacientes foi de 8,39±10,9 dias, e a causa mais frequente de inter- namento foi devido a pós-operatório por diversas causas, somando 148 indivíduos (74%). Dentro os pacientes, 72 (36%) apresenta- ram alguma complicação. Além disso, 13 casos (6,5%) evoluíram para óbito. Conclusão: Estudos disponíveis sobre alvos de gli- cose em pacientes críticos das unidades de terapia intensiva apresentam difícil interpretação devido às diferenças subs- tanciais no grupo de populações e aos protocolos de gestão de pacientes utilizados em vários centros. Todavia, a prevalência da hiperglicemia de estresse encontrada nesta amostra é se- melhante à de outras casuísticas estudadas. O índice eleva- do de complicações enfatiza a necessidade de padronização nos critérios para diagnóstico e tratamento da hiperglicemia de estresse objetivando melhor prognóstico desses pacientes independentemente da causa do internamento.

Objective: To evaluate the prevalence and management of stress hyperglycemia in patients hospitalized in anintensive care unit. Methods: Retrospective study, carried out from January to June 2018. Data were obtained from 582 electronic medical records, considering glycemic values during hospitalization, existence of previous history of Diabetes Mellitus, causes of hospitalization, length of stay in the intensive care unit, presence of complications during hospitalization, and behavior used in case of stress hyper- glycemia. Results: Of the 582 patients admitted in the ICU, 579 had their glycemia indicated in the charts analyzed: 341 (58,9%) had hyperglycemia in a certain moment of hospitalization, with stress hyperglycemia being present in 200 patients (35%). The average duration of hospitalization of these patients was 8,39 ± 10,9 days, and the most frequent cause of hospitalization was postoperative for various causes, totaling 148 individuals (74%). Of the patients, 72 (36%) presented some type of complication and 13 patients (6,5%) died. Conclusion: Available studies on glucose targets in critical intensive care unit patients are difficult to be interpre- ted because of substantial differences in the study populations and of patient management protocols used at various centers. However, the prevalence of stress hyperglycemia found in this sample is similar to that of other study groups. The high com- plication rate emphasizes the need for standardization of the criteria for diagnosis and treatment of stress hyperglycemia aiming at a better prognosis of these patients regardless of the cause of hospitalization.

Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato.

Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.

RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant.

BACKGROUND: In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. METHODS: We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. RESULTS: Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). CONCLUSION: Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

Intracellular proliferation of clover yellow vein virus is unaffected by the recessive resistance gene cyv1 of Pisum sativum.

The pea cyv1 gene is a yet-to-be-identified recessive resistance gene that inhibits the infection of clover yellow vein virus (ClYVV). Previous studies confirmed that the cell-to-cell movement of ClYVV is inhibited in cyv1-carrying pea plants; however, the effect of cyv1 on viral replication remains unknown. In this study, we developed a new pea protoplast transfection method to investigate ClYVV propagation at the single-cell level. Using this method, we revealed that ClYVV accumulates to similar levels in both ClYVV-susceptible and cyv1-carrying pea protoplasts. Thus, the cyv1-mediated resistance would not suppress intracellular ClYVV replication.

Trifluoromethyl Dihydrothiazine-Based ß-Secretase (BACE1) Inhibitors with Robust Central ß-Amyloid Reduction and Minimal Covalent Binding Burden.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as ß-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aß reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.

A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.

Structure-Based Design of Selective ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitors: Targeting the Flap to Gain Selectivity over BACE2.

BACE1 inhibitors hold potential as agents in disease-modifying treatment for Alzheimer's disease. BACE2 cleaves the melanocyte protein PMEL in pigment cells of the skin and eye, generating melanin pigments. This role of BACE2 implies that nonselective and chronic inhibition of BACE1 may cause side effects derived from BACE2. Herein, we describe the discovery of potent and selective BACE1 inhibitors using structure-based drug design. We targeted the flap region, where the shape and flexibility differ between these enzymes. Analysis of the cocrystal structures of an initial lead 8 prompted us to incorporate spirocycles followed by its fine-tuning, culminating in highly selective compounds 21 and 22. The structures of 22 bound to BACE1 and BACE2 revealed that a relatively high energetic penalty in the flap of the 22-bound BACE2 structure may cause a loss in BACE2 potency, thereby leading to its high selectivity. These findings and insights should contribute to responding to the challenges in exploring selective BACE1 inhibitors.

Recessive Resistance Governed by a Major Quantitative Trait Locus Restricts Clover Yellow Vein Virus in Mechanically but Not Graft-Inoculated Cultivated Soybeans.

Clover yellow vein virus (ClYVV) infects and causes disease in legume plants. However, here, we found that ClYVV isolate No. 30 (ClYVV-No.30) inefficiently multiplied or spread via cell-to-cell movement in mechanically inoculated leaves of a dozen soybean (Glycine max) cultivars and resulted in failure to spread systemically. Soybean plants also had a similar resistance phenotype against additional ClYVV isolates. In contrast, all but one of 24 tested accessions of wild soybeans (G. soja) were susceptible to ClYVV-No.30. Graft inoculation of cultivated soybean TK780 with ClYVV-No.30-infected wild soybean B01167 scion resulted in systemic infection of the cultivated soybean rootstock. This suggests that, upon mechanical inoculation, the cultivated soybean inhibits ClYVV-No.30, at infection steps prior to the systemic spread of the virus, via vascular systems. Systemic infection of all F1 plants from crossing between TK780 and B01167 and of 68 of 76 F2 plants with ClYVV-No.30 indicated recessive inheritance of the resistance. Further genetic analysis using 64 recombinant inbred lines between TK780 and B01167 detected one major quantitative trait locus, designated d-cv, for the resistance that was positioned in the linkage group D1b (chromosome 2). The mapped region on soybean genome suggests that d-cv is not an allele of the known resistance genes against soybean mosaic virus.

Discovery of Potent and Centrally Active 6-Substituted 5-Fluoro-1,3-dihydro-oxazine ß-Secretase (BACE1) Inhibitors via Active Conformation Stabilization.

ß-Secretase (BACE1) has an essential role in the production of amyloid ß peptides that accumulate in patients with Alzheimer's disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aß reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

Trade-Offs for Viruses in Overcoming Innate Immunities in Plants.

Plants recognize viral infection via an immune receptor, i.e., nucleotide-binding site (NB)-leucine-rich repeat (LRR) proteins. Another immune receptor, receptor-like kinase proteins, which share an LRR domain with NB-LRRs, perceive conserved molecules of pathogens called pathogen- or microbe-associated molecular patterns, but NB-LRRs generally perceive particular viral proteins. As viruses can evolve more rapidly than the host immune system, how do plant immune systems, which rely on the perception of proteins, remain effective? Viral adaptive evolution may be controlled by penalties that result from mutations in viral proteins that are perceived by NB-LRRs. Our recent studies in pea (Pisum sativum) suggest a penalty of increased susceptibility to another immune system. When a viral protein mutates to evade one immune system, the virus with the mutated protein becomes more susceptible to another. Such antagonistic pleiotropy of a viral protein by two independent plant immune systems may have precedents. Plants may rely on pairs of immune systems to constrain adaptive evolution by viruses and thereby maintain durable antiviral immunity. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC 4.0 International license .

P3N-PIPO, a Frameshift Product from the P3 Gene, Pleiotropically Determines the Virulence of Clover Yellow Vein Virus in both Resistant and Susceptible Peas.

UNLABELLED: Peas carrying the cyv1 recessive resistance gene are resistant to clover yellow vein virus (ClYVV) isolates No.30 (Cl-No.30) and 90-1 (Cl-90-1) but can be infected by a derivative of Cl-90-1 (Cl-90-1 Br2). The main determinant for the breaking of cyv1 resistance by Cl-90-1 Br2 is P3N-PIPO produced from the P3 gene via transcriptional slippage, and the higher level of P3N-PIPO produced by Cl-90-1 Br2 than by Cl-No.30 contributes to the breaking of resistance. Here we show that P3N-PIPO is also a major virulence determinant in susceptible peas that possess another resistance gene, Cyn1, which does not inhibit systemic infection with ClYVV but causes hypersensitive reaction-like lethal systemic cell death. We previously assumed that the susceptible pea cultivar PI 226564 has a weak allele of Cyn1 Cl-No.30 did not induce cell death, but Cl-90-1 Br2 killed the plants. Our results suggest that P3N-PIPO is recognized by Cyn1 and induces cell death. Unexpectedly, heterologously strongly expressed P3N-PIPO of Cl-No.30 appears to be recognized by Cyn1 in PI 226564. The level of P3N-PIPO accumulation from the P3 gene of Cl-No.30 was significantly lower than that of Cl-90-1 Br2 in a Nicotiana benthamiana transient assay. Therefore, Cyn1-mediated cell death also appears to be determined by the level of P3N-PIPO. The more efficiently a ClYVV isolate broke cyv1 resistance, the more it induced cell death systemically (resulting in a loss of the environment for virus accumulation) in susceptible peas carrying Cyn1, suggesting that antagonistic pleiotropy of P3N-PIPO controls the resistance breaking of ClYVV. IMPORTANCE: Control of plant viral disease has relied on the use of resistant cultivars; however, emerging mutant viruses have broken many types of resistance. Recently, we revealed that Cl-90-1 Br2 breaks the recessive resistance conferred by cyv1, mainly by accumulating a higher level of P3N-PIPO than that of the nonbreaking isolate Cl-No.30. Here we show that a susceptible pea line recognized the increased amount of P3N-PIPO produced by Cl-90-1 Br2 and activated the salicylic acid-mediated defense pathway, inducing lethal systemic cell death. We found a gradation of virulence among ClYVV isolates in a cyv1-carrying pea line and two susceptible pea lines. This study suggests a trade-off between breaking of recessive resistance (cyv1) and host viability; the latter is presumably regulated by the dominant Cyn1 gene, which may impose evolutionary constraints upon P3N-PIPO for overcoming resistance. We propose a working model of the host strategy to sustain the durability of resistance and control fast-evolving viruses.