The plant disease triangle facing climate change: a molecular perspective.

Variations in climate conditions can dramatically affect plant health and the generation of climate-resilient crops is imperative to food security. In addition to directly affecting plants, it is predicted that more severe climate conditions will also result in greater biotic stresses. Recent studies have identified climate-sensitive molecular pathways that can result in plants being more susceptible to infection under unfavorable conditions. Here, we review how expected changes in climate will impact plant-pathogen interactions, with a focus on mechanisms regulating plant immunity and microbial virulence strategies. We highlight the complex interactions between abiotic and biotic stresses with the goal of identifying components and/or pathways that are promising targets for genetic engineering to enhance adaptation and strengthen resilience in dynamically changing environments.

Extracellular niche establishment by plant pathogens.

The plant extracellular space, referred to as the apoplast, is inhabited by a variety of microorganisms. Reflecting the crucial nature of this compartment, both plants and microorganisms seek to control, exploit and respond to its composition. Upon sensing the apoplastic environment, pathogens activate virulence programmes, including the delivery of effectors with well-established roles in suppressing plant immunity. We posit that another key and foundational role of effectors is niche establishment - specifically, the manipulation of plant physiological processes to enrich the apoplast in water and nutritive metabolites. Facets of plant immunity counteract niche establishment by restricting water, nutrients and signals for virulence activation. The complex competition to control and, in the case of pathogens, exploit the apoplast provides remarkable insights into the nature of virulence, host susceptibility, host defence and, ultimately, the origin of phytopathogenesis. This novel framework focuses on the ecology of a microbial niche and highlights areas of future research on plant-microorganism interactions.

Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.

NanoViromics: long-read sequencing of dsRNA for plant virus and viroid rapid detection.

There is a global need for identifying viral pathogens, as well as for providing certified clean plant materials, in order to limit the spread of viral diseases. A key component of management programs for viral-like diseases is having a diagnostic tool that is quick, reliable, inexpensive, and easy to use. We have developed and validated a dsRNA-based nanopore sequencing protocol as a reliable method for detecting viruses and viroids in grapevines. We compared our method, which we term direct-cDNA sequencing from dsRNA (dsRNAcD), to direct RNA sequencing from rRNA-depleted total RNA (rdTotalRNA), and found that it provided more viral reads from infected samples. Indeed, dsRNAcD was able to detect all of the viruses and viroids detected using Illumina MiSeq sequencing (dsRNA-MiSeq). Furthermore, dsRNAcD sequencing was also able to detect low-abundance viruses that rdTotalRNA sequencing failed to detect. Additionally, rdTotalRNA sequencing resulted in a false-positive viroid identification due to the misannotation of a host-driven read. Two taxonomic classification workflows, DIAMOND & MEGAN (DIA & MEG) and Centrifuge & Recentrifuge (Cent & Rec), were also evaluated for quick and accurate read classification. Although the results from both workflows were similar, we identified pros and cons for both workflows. Our study shows that dsRNAcD sequencing and the proposed data analysis workflows are suitable for consistent detection of viruses and viroids, particularly in grapevines where mixed viral infections are common.

Evidence of the Involvement of Nematostoma parasiticum in Herpotrichia Needle Browning of Abies balsamea.

Herpotrichia needle browning (HNB) is a disease that affects several species of fir trees in Europe and North America. HNB was first described by Hartig in 1884, who isolated a fungal pathogenic agent identified as responsible for the disease. This fungus was later named Herpotrichia parasitica but is currently named Nematostoma parasiticum. However, the identity of the pathogens causing HNB is regularly questioned and, to date, the true causal agent of this disease has not been definitely established. The present study aimed to identify the fungal populations present in needles of Christmas fir trees (Abies balsamea) and to correlate them with needle health status using robust molecular methods. PCR primers specific to N. parasiticum allowed detection of the presence of this fungus in DNA samples from symptomatic needles. Furthermore, high-throughput sequencing (Illumina MiSeq) clearly showed that N. parasiticum was associated with symptomatic needles. However, high-throughput sequencing results revealed that the presence of other species such as Sydowia polyspora and Rhizoctonia sp. may also correlate with the development of HNB. A diagnostic tool, based on quantitative PCR using a probe, was then developed to detect and quantify N. parasiticum in DNA samples. The efficacy of this molecular approach was validated through the detection of the pathogenic agent in symptomatic needle samples as well as in nonsymptomatic needles collected in trees affected by HNB. In contrast, N. parasiticum could not be found in needles from healthy trees. The present study argues for the importance of N. parasiticum in causing HNB symptoms.

Light prevents pathogen-induced aqueous microenvironments via potentiation of salicylic acid signaling.

Many plant pathogens induce water-soaked lesions in infected tissues. In the case of Pseudomonas syringae (Pst), water-soaking effectors stimulate abscisic acid (ABA) production and signaling, resulting in stomatal closure. This reduces transpiration, increases water accumulation, and induces an apoplastic microenvironment favorable for bacterial growth. Stomata are sensitive to environmental conditions, including light. Here, we show that a period of darkness is required for water-soaking, and that a constant light regime abrogates stomatal closure by Pst. We find that constant light induces resistance to Pst, and that this effect requires salicylic acid (SA). Constant light did not alter effector-induced accumulation of ABA, but induced greater SA production, promoting stomatal opening despite the presence of ABA. Furthermore, application of a SA analog was sufficient to prevent pathogen-induced stomatal closure and water-soaking. Our results suggest potential approaches for interfering with a common virulence strategy, as well as providing a physiological mechanism by which SA functions in defense against pathogens.

A Jasmonic Acid-Related Mechanism Affects ARGONAUTE5 Expression and Antiviral Defense Against Potato Virus X in Arabidopsis thaliana.

During virus infection, Argonaute (AGO) proteins bind to Dicer-produced virus small interfering RNAs and target viral RNA based on sequence complementarity, thereby limiting virus proliferation. The Arabidopsis AGO2 protein is important for resistance to multiple viruses, including potato virus X (PVX). In addition, AGO5 is important in systemic defense against PVX. Normally AGO5 is expressed only in reproductive tissues, and its induction by virus infection is thought to be important for its participation in antiviral defense. However, it is unclear what mechanisms induce AGO5 expression in response to virus infection. Here, we show that dde2-2, a mutant compromised in jasmonic acid (JA) biosynthesis, displays constitutive upregulation of AGO5. This mutant also showed increased resistance to PVX and this resistance was dependent on a functional AGO5 gene. Furthermore, methyl jasmonate treatment ablated AGO5 expression in leaves during virus infection and resulted in increased susceptibility to virus. Our results further support a role for AGO5 in antiviral RNA silencing and a negative regulation by JA, a plant hormone associated with defense against plant-feeding arthropods, which are often the vectors of plant viruses. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Osmotic Demyelination Syndrome in a Patient With Tremors.

Osmotic demyelination syndrome is a neurological disorder caused by damage to the myelin sheath of brain cells secondary to rapid correction of hyponatremia. Clinical features are variable depending on the location of demyelination, with diagnosis confirmed by MRI. Once diagnosed, treatment is supportive. We present a 49-year-old female recently discharged from an outside hospital who presented with symptoms of tremors, ataxia, slurred speech, and confusion. The patient was diagnosed with osmotic demyelination syndrome based on the classic trident sign on MRI imaging. A review of her records showed rapid correction of serum sodium during her initial hospital visit.

N and N'-mediated recognition confers resistance to tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that overcomes the Tm-2 2 resistance gene used in commercial tomato plants to protect against tobamoviruses. In this article, we show that ToBRFV is recognised through its P50 replicase fragment by the resistance gene N in N. tabacum , which triggers a hypersensitive response (HR). We also demonstrate that the N' gene provides protection against ToBRFV through recognition of the viral coat protein without triggering a typical HR in N. tabacum .

Evolutionarily conserved bacterial effectors hijack abscisic acid signaling to induce an aqueous environment in the apoplast.

High atmospheric humidity levels profoundly impact host-pathogen interactions in plants by enabling the establishment of an aqueous living space that benefits pathogens. The effectors HopM1 and AvrE1 of the bacterial pathogen Pseudomonas syringae have been shown to induce an aqueous apoplast under such conditions. However, the mechanisms by which this happens remain unknown. Here, we show that HopM1 and AvrE1 work redundantly to establish an aqueous living space by inducing a major reprogramming of the Arabidopsis thaliana transcriptome landscape. These effectors induce a strong abscisic acid (ABA) signature, which promotes stomatal closure, resulting in reduced leaf transpiration and water-soaking lesions. Furthermore, these effectors preferentially increase ABA accumulation in guard cells, which control stomatal aperture. Notably, a guard-cell-specific ABA transporter, ABCG40, is necessary for HopM1 induction of water-soaking lesions. This study provides molecular insights into a chain of events of stomatal manipulation that create an ideal microenvironment to facilitate infection.

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus-plant interactions, as well as the characterization of viral RNA modifications.

What does it take to be antiviral? An Argonaute-centered perspective on plant antiviral defense.

RNA silencing is a major mechanism of constitutive antiviral defense in plants, mediated by a number of proteins, including the Dicer-like (DCL) and Argonaute (AGO) endoribonucleases. Both DCL and AGO protein families comprise multiple members. In particular, the AGO protein family has expanded considerably in different plant lineages, with different family members having specialized functions. Although the general mode of action of AGO proteins is well established, the properties that make different AGO proteins more or less efficient at targeting viruses are less well understood. In this report, we review methodologies used to study AGO antiviral activity and current knowledge about which AGO family members are involved in antiviral defense. In addition, we discuss what is known about the different properties of AGO proteins thought to be associated with this function.

Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite.

Codiaeum variegatum (common name, garden croton) is an ornamental plant grown for its bright yellow variegated leaf morphology. Two C. variegatum plants with upward leaf curling and vein swelling symptoms were collected in Faisalabad, Pakistan. Sequencing of clones obtained by PCR amplification with specific primers showed one plant infected with the monopartite begomoviruses pedilanthus leaf curl virus (PeLCV) and papaya leaf curl virus (PaLCuV) and the other to be infected with only PeLCV. Both plants also harboured a betasatellite that was distinct from all previously identified betasatellites, for which the name "codiaeum leaf curl betasatellite" (CoLCuB) is proposed. This is the first identification of a begomovirus and an associated betasatellite infecting C. variegatum in Pakistan. Both PeLCV and PaLCuV cause problems in a number of crop plants, and C. variegatum may act as a reservoir for these agriculturally important viruses. The precise impact and geographical distribution of the newly identified CoLCuB will be investigated.

ARGONAUTE5 Represses Age-Dependent Induction of Flowering through Physical and Functional Interaction with miR156 in Arabidopsis.

Flowering time is a finely tuned process in plants, in part controlled by the age-regulated microRNA156 (miR156), which functions by suppressing the transcripts of SQUAMOSA-PROMOTER BINDING LIKE (SPL) transcription factors. ARGONAUTE (AGO) proteins are essential effectors of miRNA-mediated gene regulation. However, which AGO(s) mediate(s) the control of flowering time remains unclear. Here, we demonstrate a role of AGO5 in controlling flowering time by modulating the expression of SPL transcription factors. We show that AGO5 interacts physically and functionally with miR156 and that ago5 mutants present an early flowering phenotype in Arabidopsis. Furthermore, in ago5 mutants, the repression of flowering caused by miR156 overexpression is largely reversed, whereas leaf morphology remains unaffected. Our results thus indicate a specific role for AGO5 in mediating miR156 activity in meristematic, but not vegetative, tissue. As such, our data suggest a spatiotemporal regulation of the miR156 aging pathway mediated through different AGO proteins in different tissues.

Natural variation in the Arabidopsis AGO2 gene is associated with susceptibility to potato virus X.

RNA silencing functions as an anti-viral defence in plants through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. Despite the importance of this mechanism, little is known about the functional consequences of variation in genes encoding RNA silencing components. The AGO2 protein has been shown to be important for defense against multiple viruses, and we investigated how naturally occurring differences in AGO2 between and within species affects its antiviral activities. We find that the AGO2 protein from Arabidopsis thaliana, but not Nicotiana benthamiana, effectively limits potato virus X (PVX). Consistent with this, we find that the A. thaliana AGO2 gene shows a high incidence of polymorphisms between accessions, with evidence of selective pressure. Using functional analyses, we identify polymorphisms that specifically affect AGO2 antiviral activity, without interfering with other AGO2-associated functions such as anti-bacterial resistance or DNA methylation. Our results suggest that viruses adapt to overcome RNA silencing in their hosts. Furthermore, they indicate that plant-virus interactions have influenced natural variation in RNA-silencing components and that the latter may be a source of genetically encoded virus resistance.

Standardized Letters of Concern and Remediation Contracts: Templates for Program Directors.

BACKGROUND: Remediation of the struggling resident is a universal phenomenon, and the majority of program directors will remediate at least 1 resident during their tenure. OBJECTIVE: The goal of this project was to create a standardized template for program directors to use at all stages of remediation. METHODS: Between 2017 and 2018, the Council of Residency Directors in Emergency Medicine (CORD-EM) Remediation Committee searched for best practices in the medical literature and compiled a survey that was e-mailed to the CORD-EM listserv. After reviewing all information, a standardized remediation contract was created, reviewed by legal counsel, and distributed to members. RESULTS: Forty-two percent (110 of 263) of program directors or assistant program directors on the CORD-EM listserv answered the initial survey and provided guidance on current remediation practices. The committee created formal and informal standard remediation contracts as both fillable templates and alterable documents. These were reviewed by CORD-EM general legal counsel and approved by the CORD-EM Board of Directors for distribution. The project took approximately 20 hours to complete over 8 months and involved a cost of $480 for legal fees. CONCLUSIONS: With program director input and legal counsel review, the CORD-EM Remediation Committee produced standardized remediation contracts, which can be used by all emergency medicine programs after comparison to local institutional policy and local legal review. This process was feasible and can be replicated by other specialties.

Alterations in cellular RNA decapping dynamics affect tomato spotted wilt virus cap snatching and infection in Arabidopsis.

RNA processing and decay pathways have important impacts on RNA viruses, particularly animal-infecting bunyaviruses, which utilize a cap-snatching mechanism to translate their mRNAs. However, their effects on plant-infecting bunyaviruses have not been investigated. The roles of mRNA degradation and non-sense-mediated decay components, including DECAPPING 2 (DCP2), EXORIBONUCLEASE 4 (XRN4), ASYMMETRIC LEAVES2 (AS2) and UP-FRAMESHIFT 1 (UPF1) were investigated in infection of Arabidopsis thaliana by several RNA viruses, including the bunyavirus, tomato spotted wilt virus (TSWV). TSWV infection on mutants with decreased or increased RNA decapping ability resulted in increased and decreased susceptibility, respectively. By contrast, these mutations had the opposite, or no, effect on RNA viruses that use different mRNA capping strategies. Consistent with this, the RNA capping efficiency of TSWV mRNA was higher in a dcp2 mutant. Furthermore, the TSWV N protein partially colocalized with RNA processing body (PB) components and altering decapping activity by heat shock or coinfection with another virus resulted in corresponding changes in TSWV accumulation. The present results indicate that TSWV infection in plants depends on its ability to snatch caps from mRNAs destined for decapping in PBs and that genetic or environmental alteration of RNA processing dynamics can affect infection outcomes.

Functional analysis of apple stem pitting virus coat protein variants.

BACKGROUND: Although the canonical function of viral coat protein (CP) is to encapsidate the viral genome, they have come to be recognized as multifunctional proteins, involved in almost every stage of the viral infection cycle. However, CP functions of Apple stem pitting virus (ASPV) has not been comprehensively documented. This study aimed to characterize the functions of ASPV CP and any functional diversification caused by sequence diversity of six ASPV CP variants and studied their biological, serological, pathogenic and viral suppressor of RNA silencing (VSR) functions. METHODS: Six ASPV CP variants that have previously been shown to belong to different subgroups were selected here to study their diversity functions. Agrobacterium mediated infiltration (Agroinfiltration) was used to express YFP-ASPV-CPs in Nicotiana. benthamiana and infect Nicotiana. occidental with PVX-ASPV-CPs in. Confocal microscopy was used to detect YFP-ASPV-CPs florescence. CPs expressed in Escherichia coli BL21 (DE3) were induced by IPTG. RESULTS: In this study, we showed that recombinant CPs expressed in Escherichia coli BL21 (DE3) had different levels of serological reactivity to three anti-ASPV antibodies used to detect ASPV. Furthermore, fusion CPs with YFP (YFP-CPs) expressed in N. benthamiana cells differed in their ability to form aggregates. We also showed that ASPV isolates that harbour these CPs induced different biological symptoms on its herbaceous host N. occidentalis. At the same time, we found that all six CPs when expressed in PVX vector showed similar VSR activity and produced similar symptoms in N. occidentalis, despite their differences in amino acids. CONCLUSIONS: Different ASPV isolates induced different symptoms in N. occidentalis, however, ASPV CP variants expressed in PVX vector showed the same symptoms in N. occidentalis plants. Also, we showed that ASPV CP variants has the same level of VSR activity, but they have different abilities to aggregate in N. benthamiana.

Cell death triggered by the P25 protein in Potato virus X-associated synergisms results from endoplasmic reticulum stress in Nicotiana benthamiana.

The synergistic interaction of Potato virus X (PVX) with a number of potyviruses results in systemic necrosis in Nicotiana spp. Previous investigations have indicated that the viral suppressor of RNA silencing (VSR) protein P25 of PVX triggers systemic necrosis in PVX-associated synergisms in a threshold-dependent manner. However, little is still known about the cellular processes that lead to this necrosis, and whether the VSR activity of P25 is involved in its elicitation. Here, we show that transient expression of P25 in the presence of VSRs from different viruses, including the helper component-proteinase (HC-Pro) of potyviruses, induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which ultimately lead to ER collapse. However, the host RNA silencing pathway was dispensable for the elicitation of cell death by P25. Confocal microscopy studies in leaf patches co-expressing P25 and HC-Pro showed dramatic alterations in ER membrane structures, which correlated with the up-regulation of bZIP60 and several ER-resident chaperones, including the ER luminal binding protein (BiP). Overexpression of BiP alleviated the cell death induced by the potexviral P25 protein when expressed together with VSRs derived from different viruses. Conversely, silencing of the UPR master regulator, bZIP60, led to an increase in cell death elicited by the P25/HC-Pro combination as well as by PVX-associated synergism. In addition to its role as a negative regulator of P25-induced cell death, UPR partially restricted PVX infection. Thus, systemic necrosis caused by PVX-associated synergistic infections is probably the effect of an unmitigated ER stress following the overaccumulation of a viral protein, P25, with ER remodelling activity.