Extensive membrane systems at the host-arbuscular mycorrhizal fungus interface.

During arbuscular mycorrhizal (AM) symbiosis, cells within the root cortex develop a matrix-filled apoplastic compartment in which differentiated AM fungal hyphae called arbuscules reside. Development of the compartment occurs rapidly, coincident with intracellular penetration and rapid branching of the fungal hypha, and it requires much of the plant cell's secretory machinery to generate the periarbuscular membrane that delimits the compartment. Despite recent advances, our understanding of the development of the periarbuscular membrane and the transfer of molecules across the symbiotic interface is limited. Here, using electron microscopy and tomography, we reveal that the periarbuscular matrix contains two types of membrane-bound compartments. We propose that one of these arises as a consequence of biogenesis of the periarbuscular membrane and may facilitate movement of molecules between symbiotic partners. Additionally, we show that the arbuscule contains massive arrays of membrane tubules located between the protoplast and the cell wall. We speculate that these tubules may provide the absorptive capacity needed for nutrient assimilation and possibly water absorption to enable rapid hyphal expansion.

A novel bi-domain plant defensin MtDef5 with potent broad-spectrum antifungal activity binds to multiple phospholipids and forms oligomers.

Defensins are cysteine-rich cationic antimicrobial peptides contributing to the innate immunity in plants. A unique gene encoding a highly cationic bi-domain defensin MtDef5 has been identified in a model legume Medicago truncatula. MtDef5 consists of two defensin domains of 50 amino acids each linked by a 7-amino acid peptide. It exhibits broad-spectrum antifungal activity against filamentous fungi at submicromolar concentrations. It rapidly permeabilizes the plasma membrane of the ascomycete fungi Fusarium graminearum and Neurospora crassa and induces accumulation of reactive oxygen species. It is internalized by these fungi, but uses spatially distinct modes of entry into these fungi. It co-localizes with cellular membranes, travels to nucleus and becomes dispersed in other subcellular locations. It binds to several membrane-resident phospholipids with preference for phosphatidylinositol monophosphates and forms oligomers. Mutations of the cationic amino acids present in the two γ-core motifs of this defensin that eliminate oligomerization also knockout its ability to induce membrane permeabilization and fungal growth arrest. MtDef5 is the first bi-domain plant defensin that exhibits potent broad-spectrum antifungal activity, recruits multiple membrane phospholipids and forms oligomers in their presence. These findings raise the possibility that MtDef5 might be useful as a novel antifungal agent in transgenic crops.

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f.

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

Plastid osmotic stress influences cell differentiation at the plant shoot apex.

The balance between proliferation and differentiation in the plant shoot apical meristem is controlled by regulatory loops involving the phytohormone cytokinin and stem cell identity genes. Concurrently, cellular differentiation in the developing shoot is coordinated with the environmental and developmental status of plastids within those cells. Here, we employ an Arabidopsis thaliana mutant exhibiting constitutive plastid osmotic stress to investigate the molecular and genetic pathways connecting plastid osmotic stress with cell differentiation at the shoot apex. msl2 msl3 mutants exhibit dramatically enlarged and deformed plastids in the shoot apical meristem, and develop a mass of callus tissue at the shoot apex. Callus production in this mutant requires the cytokinin receptor AHK2 and is characterized by increased cytokinin levels, downregulation of cytokinin signaling inhibitors ARR7 and ARR15, and induction of the stem cell identity gene WUSCHEL Furthermore, plastid stress-induced apical callus production requires elevated plastidic reactive oxygen species, ABA biosynthesis, the retrograde signaling protein GUN1, and ABI4. These results are consistent with a model wherein the cytokinin/WUS pathway and retrograde signaling control cell differentiation at the shoot apex.

Transgenic soya bean seeds accumulating ß-carotene exhibit the collateral enhancements of oleate and protein content traits.

Transgenic soya bean (Glycine max) plants overexpressing a seed-specific bacterial phytoene synthase gene from Pantoea ananatis modified to target to plastids accumulated 845 µg ß carotene g(-1) dry seed weight with a desirable 12:1 ratio of ß to α. The ß carotene accumulating seeds exhibited a shift in oil composition increasing oleic acid with a concomitant decrease in linoleic acid and an increase in seed protein content by at least 4% (w/w). Elevated ß-carotene accumulating soya bean cotyledons contain 40% the amount of abscisic acid compared to nontransgenic cotyledons. Proteomic and nontargeted metabolomic analysis of the mid-maturation ß-carotene cotyledons compared to the nontransgenic did not reveal any significant differences that would account for the altered phenotypes of both elevated oleate and protein content. Transcriptomic analysis, confirmed by RT-PCR, revealed a number of significant differences in ABA-responsive transcripton factor gene expression in the crtB transgenics compared to nontransgenic cotyledons of the same maturation stage. The altered seed composition traits seem to be attributed to altered ABA hormone levels varying transcription factor expression. The elevated ß-carotene, oleic acid and protein traits in the ß-carotene soya beans confer a substantial additive nutritional quality to soya beans.

Immunolocalization of glutathione biosynthesis enzymes in Arabidopsis thaliana.

In plants, glutathione serves as a versatile redox buffer and cellular protective compound against a range of biotic and abiotic stresses. Glutathione production involves glutamate-cysteine ligase (GCL), the redox-regulated limiting enzyme of the pathway, and glutathione synthetase (GS). Because the sub-cellular and sub-organellar localization of these enzymes will have an impact on metabolism, here we examine the localization of GCL and GS in the leaves of Arabidopsis thaliana. Immuno-electron microscopy of leaf cells indicates localization of GCL primarily to the chloroplast with GS found in both the chloroplast and cytosol. Detailed examination of the localization of both enzymes within chloroplasts was performed using fractionation followed by immunoblot analysis and indicates that GCL and GS are found in the stroma. The localization of these enzymes to the stroma of chloroplasts has implications for the redox-regulation of GCL and plant glutathione biosynthesis.

Reproductive Toxicity and Life History Study of Silver Nanoparticle Effect, Uptake and Transport in Arabidopsis thaliana.

Concerns about nanotechnology have prompted studies on how the release of these engineered nanoparticles impact our environment. Herein, the impact of 20 nm silver nanoparticles (AgNPs) on the life history traits of Arabidopsis thaliana was studied in both above- and below-ground parts, at macroscopic and microscopic scales. Both gross phenotypes (in contrast to microscopic phenotypes) and routes of transport and accumulation were investigated from roots to shoots. Wild type Arabidopsis growing in soil, regularly irrigated with 75 µg/L of AgNPs, did not show any obvious morphological change. However, their vegetative development was prolonged by two to three days and their reproductive growth shortened by three to four days. In addition, the germination rates of offspring decreased drastically over three generations. These findings confirmed that AgNPs induce abiotic stress and cause reproductive toxicity in Arabidopsis. To trace transport of AgNPs, this study also included an Arabidopsis reporter line genetically transformed with a green fluorescent protein and grown in an optical transparent medium with 75 µg/L AgNPs. AgNPs followed three routes: (1) At seven days after planting (DAP) at S1.0 (stages defined by Boyes et al. 2001 [41]), AgNPs attached to the surface of primary roots and then entered their root tips; (2) At 14 DAP at S1.04, as primary roots grew longer, AgNPs gradually moved into roots and entered new lateral root primordia and root hairs; (3) At 17 DAP at S1.06 when the Arabidopsis root system had developed multiple lateral roots, AgNPs were present in vascular tissue and throughout the whole plant from root to shoot. In some cases, if cotyledons of the Arabidopsis seedlings were immersed in melted transparent medium, then AgNPs were taken up by and accumulated in stomatal guard cells. These findings in Arabidopsis are the first to document specific routes and rates of AgNP uptake in vivo and in situ.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA.

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

A dual-targeted soybean protein is involved in Bradyrhizobium japonicum infection of soybean root hair and cortical cells.

The symbiotic interaction between legumes and soil bacteria (e.g., soybean [Glycine max L.] and Bradyrhizobium japonicum]) leads to the development of a new root organ, the nodule, where bacteria differentiate into bacteroids that fix atmospheric nitrogen for assimilation by the plant host. In exchange, the host plant provides a steady carbon supply to the bacteroids. This carbon can be stored within the bacteroids in the form of poly-3-hydroxybutyrate granules. The formation of this symbiosis requires communication between both partners to regulate the balance between nitrogen fixation and carbon utilization. In the present study, we describe the soybean gene GmNMNa that is specifically expressed during the infection of soybean cells by B. japonicum. GmNMNa encodes a protein of unknown function. The GmNMNa protein was localized to the nucleolus and also to the mitochondria. Silencing of GmNMNa expression resulted in reduced nodulation, a reduction in the number of bacteroids per infected cell in the nodule, and a clear reduction in the accumulation of poly-3-hydroxybutyrate in the bacteroids. Our results highlight the role of the soybean GmNMNa gene in regulating symbiotic bacterial infection, potentially through the regulation of the accumulation of carbon reserves.

Insights into the complex 3-D architecture of thylakoid membranes in unicellular cyanobacterium Cyanothece sp. ATCC 51142.

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is left-handed in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.

Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography.

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.

A member of the highly conserved FWL (tomato FW2.2-like) gene family is essential for soybean nodule organogenesis.

A soybean homolog of the tomato FW2.2 gene, here named GmFWL1 (Glycine max FW2.2-like 1), was found to respond strongly to inoculation with the nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum. In tomato, the FW2.2 gene is hypothesized to control 30% of the variance in fruit weight by negatively regulating cell division. In the present study, the induction of GmFWL1 expression in root hair cells and nodules in response to B. japonicum inoculation was documented using quantitative RT-PCR and transcriptional fusions to both beta-glucuronidase (GUS) and green fluorescent protein (GFP). RNAi-mediated silencing of GmFWL1 expression resulted in a significant reduction in nodule number, with a concomitant reduction in nuclear size and changes in chromatin structure. The reduction in nuclear size is probably due to a change in DNA heterochromatinization, as the ploidy level of wild-type and RNAi-silenced nodule cells was similar. GmFWL1 was localized to the plasma membrane. The data suggest that GmFWL1 probably acts indirectly, perhaps through a cellular cascade, to affect chromatin structure/nuclei architecture. As previously proposed in tomato, this function may be a result of effects on plant cell division.

GS52 ecto-apyrase plays a critical role during soybean nodulation.

Apyrases are non-energy-coupled nucleotide phosphohydrolases that hydrolyze nucleoside triphosphates and nucleoside diphosphates to nucleoside monophosphates and orthophosphates. GS52, a soybean (Glycine soja) ecto-apyrase, was previously shown to be induced very early in response to inoculation with the symbiotic bacterium Bradyrhizobium japonicum. Overexpression of the GS52 ecto-apyrase in Lotus japonicus increased the level of rhizobial infection and enhanced nodulation. These data suggest a critical role for the GS52 ecto-apyrase during nodulation. To further investigate the role of GS52 during nodulation, we used RNA interference to silence GS52 expression in soybean (Glycine max) roots using Agrobacterium rhizogenes-mediated root transformation. Transcript levels of GS52 were significantly reduced in GS52 silenced roots and these roots exhibited reduced numbers of mature nodules. Development of the nodule primordium and subsequent nodule maturation was significantly suppressed in GS52 silenced roots. Transmission electron micrographs of GS52 silenced root nodules showed that early senescence and infected cortical cells were devoid of symbiosome-containing bacteroids. Application of exogenous adenosine diphosphate to silenced GS52 roots restored nodule development. Restored nodules contained bacteroids, thus indicating that extracellular adenosine diphosphate is important during nodulation. These results clearly suggest that GS52 ecto-apyrase catalytic activity is critical for the early B. japonicum infection process, initiation of nodule primordium development, and subsequent nodule organogenesis in soybean.

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability.

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes (AtLCB2a and AtLCB2b) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.

Fluorescent protein applications in plants.

Study of plant cell biology has benefited tremendously from the use of fluorescent proteins (FPs). Development of well-established techniques in genetics, by transient expression or by Agrobacterium-mediated plant cell transformation, makes it possible to readily create material for imaging molecules tagged with FPs. Confocal microscopy of FPs is routine and, in highly scattering tissues, multiphoton microscopy improves deep imaging. The abundance of autofluorescent compounds in plants in some cases potentially interferes with FP signals, but spectral imaging is an effective tool in unmixing overlapping signals. This approach allows separate detection of DsRed and chlorophyll, DsRed and GFP, and green fluorescent protein (GFP) and yellow fluorescent protein (YFP). FPs have been targeted to most plant organelles. Free (untargeted) FPs in plant cells are not only cytoplasmic, but also go into the nucleus due to their small size. FP fluorescence is potentially unstable in acidic vacuoles. FPs have been targeted to novel compartments, including protein storage vacuoles in seeds. Endoplasmic reticulum (ER)-targeted GFP has identified novel inclusion bodies that are surprisingly dynamic. FP-tagged Rab GTPases have allowed documentation of the dynamics of membrane trafficking. Investigation of virus infections has progressed significantly with the aid of FP-tagged virus proteins. Advanced techniques are giving plant scientists the ability to quantitatively analyze the behavior of FP-tagged proteins. Fluorescence lifetime microscopy is becoming the method of choice for fluorescence resonance energy transfer (FRET) analysis of FP-tagged proteins. Fluorescence correlation spectroscopy (FCS) of FPs provides information on molecular diffusion and intermolecular interactions. Use of FPs in elucidating the behavior of plant cells has a bright future.

Developing a new interdisciplinary lab course for undergraduate and graduate students: Plant cells and proteins.

Studies of protein function increasingly use multifaceted approaches that span disciplines including recombinant DNA technology, cell biology, and analytical biochemistry. These studies rely on sophisticated equipment and methodologies including confocal fluorescence microscopy, mass spectrometry, and X-ray crystallography that are beyond the scope of traditional laboratory courses. To equip the advanced undergraduate and beginning graduate students with an enabling base of knowledge and initial experience with advanced protein research methodologies, a laboratory course entitled Plant Cells and Proteins was developed in a partnership between Washington University and the Donald Danforth Plant Science Center in St. Louis. In this one semester course, 10-12 students obtain hands-on experience with plant tissue culture, gene transformation, subcellular localization of fluorescent recombinant proteins using confocal microscopy, purification of affinity-tagged recombinant proteins, isolation of total protein extracts, enzymatic assays, one- and two-dimensional gel electrophoresis, MALDI-TOF and ESI-Q-TOF mass spectrometry, protein crystallization, and X-ray diffraction. The course is taught as a series of modules, each led by an expert researcher. Students are evaluated based on a series of graded written reports and tests of their mastery of key concepts, interpretations, and the limitations of the experimental methods.

Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen, phosphorus and potassium deficiency.

Plant root sensing and adaptation to changes in the nutrient status of soils is vital for long-term productivity and growth. Reactive oxygen species (ROS) have been shown to play a role in root response to potassium deprivation. To determine the role of ROS in plant response to nitrogen and phosphorus deficiency, studies were conducted using wild-type Arabidopsis and several root hair mutants. The expression of several nutrient-responsive genes was determined by Northern blot, and ROS were quantified and localized in roots. The monitored genes varied in intensity and timing of expression depending on which nutrient was deficient. In response to nutrient deprivation, ROS concentrations increased in specific regions of the Arabidopsis root. Changes in ROS localization in Arabidopsis and in a set of root hair mutants suggest that the root hair cells are important for response to nitrogen and potassium. In contrast, the response to phosphorus deprivation occurs in the cortex where an increase in ROS was measured. Based on these results, we put forward the hypothesis that root hair cells in Arabidopsis contain a sensing system for nitrogen and potassium deprivation.

Nematode-induced changes of transporter gene expression in Arabidopsis roots.

Root-knot plant-parasitic nematodes (Meloidogyne spp.) account for much of the damage inflicted to plants by nematodes. The feeding sites of these nematodes consist of "giant" cells, which have characteristics of transfer cells found in other parts of plants. Increased transport activity across the plasma membrane is a hallmark of transfer cells, and giant cells provide nutrition for nematodes; therefore, we initiated a study to identify the transport processes that contribute to the development and function of nematode-induced feeding sites. The study was conducted over a 4-week period, during which time the large changes in the development of giant cells were documented. The Arabidopsis ATH1 GeneChip was used to identify the many transporter genes that were regulated by nematode infestation. Expression of 50 transporter genes from 18 different gene families was significantly changed upon nematode infestation. Sixteen transporter genes were studied in more detail using real-time reverse-transcriptase polymerase chain reaction to determine transcript abundance in nematode-induced galls that contain giant cells and uninfested regions of the root. Certain genes were expressed primarily in galls whereas others were expressed primarily in the uninfested regions of the root, and a third group was expressed evenly throughout the root. Multiple transport processes are regulated and these may play important roles in nematode feeding-site establishment and maintenance.

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.