A novel cellular factor of Nicotiana benthamiana susceptibility to tobamovirus infection

Viral infection, which entails synthesis of viral proteins and active reproduction of the viral genome, effects significant changes in the functions of many intracellular systems in plants. Along with these processes, a virus has to suppress cellular defense to create favorable conditions for its successful systemic spread in a plant. The virus exploits various cellular factors of a permissive host modulating its metabolism as well as local and systemic transport of macromolecules and photoassimilates. The Nicotiana benthamiana stress-induced gene encoding Kunitz peptidase inhibitor-like protein (KPILP) has recently been shown to be involved in chloroplast retrograde signaling regulation and stimulation of intercellular transport of macromolecules. In this paper we demonstrate the key role of KPILP in the development of tobamovius infection. Systemic infection of N. benthamiana plants with tobacco mosaic virus (TMV) or the closely related crucifer-infecting tobamovirus (crTMV) induces a drastic increase in KPILP mRNA accumulation. KPILP knockdown significantly reduces the efficiency of TMV and crTMV intercellular transport and reproduction. Plants with KPILP silencing become partially resistant to tobamovirus infection. Therefore, KPILP could be regarded as a novel proviral factor in the development of TMV and crTMV infection in N. benthamiana plants

Tobamovirus 3′-Terminal Gene Overlap May be a Mechanism for within-Host Fitness Improvement

Overlapping genes (OGs) are a universal phenomenon in all kingdoms, and viruses display a high content of OGs combined with a high rate of evolution. It is believed that the mechanism of gene overlap is based on overprinting of an existing gene. OGs help virus genes compress a maximum amount of information into short sequences, conferring viral proteins with novel features and thereby increasing their within-host fitness. Analysis of tobamovirus 3′-terminal genes reveals at least two modes of OG organization and mechanisms of interaction with the host. Originally isolated from Solanaceae species, viruses (referred to as Solanaceae-infecting) such as tobacco mosaic virus do not show 3′-terminal overlap between movement protein (MP) and coat protein (CP) genes but do contain open reading frame 6 (ORF6), which overlaps with both genes. Conversely, tobamoviruses, originally isolated from Brassicaceae species (referred to as Brassicaceae-infecting) and also able to infect Solanaceae plants, have no ORF6 but are characterized by overlapping MP and CP genes. Our analysis showed that the MP/CP overlap of Brassicaceae-infecting tobamoviruses results in the following: (i) genome compression and strengthening of subgenomic promoters; (ii) CP gene early expression directly from genomic and dicistronic MP subgenomic mRNA using an internal ribosome entry site (IRES) and a stable hairpin structure in the overlapping region; (iii) loss of ORF6, which influences the symptomatology of Solanaceae-infecting tobamoviruses; and (iv) acquisition of an IRES polypurine-rich region encoding an MP nuclear localization signal. We believe that MP/CP gene overlap may constitute a mechanism for host range expansion and virus adjustment to Brassicaceae plants

Methanol in Plant Life

Until recently, plant-emitted methanol was considered a biochemical by-product, but studies in the last decade have revealed its role as a signal molecule in plant-plant and plant-animal communication. Moreover, methanol participates in metabolic biochemical processes during growth and development. The purpose of this review is to determine the impact of methanol on the growth and immunity of plants. Plants generate methanol in the reaction of the demethylation of macromolecules including DNA and proteins, but the main source of plant-derived methanol is cell wall pectins, which are demethylesterified by pectin methylesterases (PMEs). Methanol emissions increase in response to mechanical wounding or other stresses due to damage of the cell wall, which is the main source of methanol production. Gaseous methanol from the wounded plant induces defense reactions in intact leaves of the same and neighboring plants, activating so-called methanol-inducible genes (MIGs) that regulate plant resistance to biotic and abiotic factors. Since PMEs are the key enzymes in methanol production, their expression increases in response to wounding, but after elimination of the stress factor effects, the plant cell should return to the original state. The amount of functional PMEs in the cell is strictly regulated at both the gene and protein levels. There is negative feedback between one of the MIGs, aldose epimerase-like protein, and PME gene transcription; moreover, the enzymatic activity of PMEs is modulated and controlled by PME inhibitors (PMEIs), which are also induced in response to pathogenic attack

Nicotiana benthamiana γ-Thionin Synthesis Is Induced in Response to Foreign Nucleus-Targeted Proteins

Pathogenic and symbiotic bacteria secrete protein factors—nucleomodulins—to affect the host cell nucleus. During evolution, plants have acquired a great variety of defense mechanisms, including the synthesis of such antimicrobial peptides (AMPs) as defensins. We have demonstrated that the transient production of a foreign protein containing nuclear localization signal (NLS) in Nicotiana benthamiana leaves leads to the increased expression of the γ-thionin (NbγThio) that belongs to the defensin group of AMPs. We hypothesized that NbγThio is induced by the nucleomodulins of pathogenic bacteria and, in particular, in response to their NLSs. We used artificial nuclear proteins based on green fluorescent protein (GFP) fused with the human prothymosin α NLS or VirE3 NLS from Agrobacterium tumefaciens as mimetics of bacterial effectors. We demonstrated that the super-production of these NLS-containing reporters in the transient expression system in N. benthamiana leaves resulted in the increase in the NbγThio mRNA level. We isolated the NbγThio gene promoter (PrγThio) and created an expression vector (PrγThio-GUS) directing GUS synthesis in agroinfiltrated leaves. The co-expression of PrγThio-GUS with 35S-GFP:NLS variants led to the significant stimulation of GUS synthesis. We concluded that NbγThio gene expression is activated in response to bacterial nucleus-targeted proteins in the cell and is regulated both at the level of transcription and post-transcription stages

CELL WALL METHANOL AS A SIGNAL IN PLANT IMMUNITY

Cell wall pectin forms a matrix around the cellulose–xyloglucan network that is composed of rhamnogalacturonan I, rhamnogalacturonan II and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids. HG is secreted in a highly methyl-esterified form and selectively de-methyl-esterified by pectin methylesterases (PMEs) during cell growth and pathogen attack. The mechanical damage that often precedes the penetration of the leaf by a pathogen promotes the activation of PME, which in turn leads to the emission of methanol (MeOH), an abundant volatile organic compound, which is quickly perceived by the intact leaves of the damaged plant and the neighboring plants. The exposure to MeOH may result in a priming effect on intact leaves, setting the stage for the within-plant and neighboring plant immunity. The emission of MeOH by a wounded plant enhances the resistance of the non-wounded, neighboring receiver plants to bacterial pathogens and promotes cell-to-cell communication that facilitates the spread of viruses in neighboring plants

Approaches to Formaldehyde Measurement: From Liquid Biological Samples to Cells and Organisms

Formaldehyde (FA) is the simplest aldehyde present both in the environment and in living organisms. FA is an extremely reactive compound capable of protein crosslinking and DNA damage. For a long time, FA was considered a “biochemical waste” and a by-product of normal cellular metabolism, but in recent decades the picture has changed. As a result, the need arose for novel instruments and approaches to monitor and measure not only environmental FA in water, cosmetics, and household products, but also in food, beverages and biological samples including cells and even organisms. Despite numerous protocols being developed for in vitro and in cellulo FA assessment, many of them have remained at the “proof-of-concept” stage. We analyze the suitability of different methods developed for non-biological objects, and present an overview of the recently developed approaches, including chemically-synthesized probes and genetically encoded FA-sensors for in cellulo and in vivo FA monitoring. We also discuss the prospects of classical methods such as chromatography and spectrophotometry, and how they have been adapted in response to the demand for precise, selective and highly sensitive evaluation of FA concentration fluctuations in biological samples. The main objectives of this review is to summarize data on the main approaches for FA content measurement in liquid biological samples, pointing out the advantages and disadvantages of each method; to report the progress in development of novel molecules suitable for application in living systems; and, finally, to discuss genetically encoded FA-sensors based on existing natural biological FA-responsive elements

Tobamovirus 3′-Terminal Gene Overlap May be a Mechanism for within-Host Fitness Improvement

Overlapping genes (OGs) are a universal phenomenon in all kingdoms, and viruses display a high content of OGs combined with a high rate of evolution. It is believed that the mechanism of gene overlap is based on overprinting of an existing gene. OGs help virus genes compress a maximum amount of information into short sequences, conferring viral proteins with novel features and thereby increasing their within-host fitness. Analysis of tobamovirus 3′-terminal genes reveals at least two modes of OG organization and mechanisms of interaction with the host. Originally isolated from Solanaceae species, viruses (referred to as Solanaceae-infecting) such as tobacco mosaic virus do not show 3′-terminal overlap between movement protein (MP) and coat protein (CP) genes but do contain open reading frame 6 (ORF6), which overlaps with both genes. Conversely, tobamoviruses, originally isolated from Brassicaceae species (referred to as Brassicaceae-infecting) and also able to infect Solanaceae plants, have no ORF6 but are characterized by overlapping MP and CP genes. Our analysis showed that the MP/CP overlap of Brassicaceae-infecting tobamoviruses results in the following: (i) genome compression and strengthening of subgenomic promoters; (ii) CP gene early expression directly from genomic and dicistronic MP subgenomic mRNA using an internal ribosome entry site (IRES) and a stable hairpin structure in the overlapping region; (iii) loss of ORF6, which influences the symptomatology of Solanaceae-infecting tobamoviruses; and (iv) acquisition of an IRES polypurine-rich region encoding an MP nuclear localization signal. We believe that MP/CP gene overlap may constitute a mechanism for host range expansion and virus adjustment to Brassicaceae plants