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This publication is intended to guide citrus growers in distinguishing the difference between citrus canker and citrus leprosis.

Callose is a 1,3-β glucan responsible for several processes in planta such as cell division, ripening of pollen mother cells, maintaining the plasmodesmata opening, and providing structure to the sieve plates. Besides the physiological roles, callose is deposited during pathogen attacks, forming papillae to prevent pathogen entrance into the tissue or plugging the sieve pores to limit the spread of phloem pathogens. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing (HLB), is a phloem-restricted pathogen whose infection leads to the production of callose in the phloem. Characterizing the dynamics of callose plugging during HLB is important for understanding the disease, but no published protocols are available for the extraction and quantification of the callose in citrus trees, and quantitative data are limited. Detection of callose by microscopy is time-consuming and expensive and does not give information about the distribution of callose in the entire plant. Here, we present a short protocol for the efficient extraction and quantification of the total callose from citrus plants. We compared different tissues from healthy and CLas-infected plants and identified an increase in callose levels in the midribs, the stems, and the fruit peduncles of infected plants. Callose levels were highest in leaves, especially midribs, compared to stems, roots, and fruit peduncles. This method could be applied to other woody plant species.

Citrus disease Huanglongbing (HLB) causes sparse (thinner) canopies due to reduced leaf and shoot biomass. Herein, we present results demonstrating the possible mechanisms behind compromised leaf growth of HLB-affected ‘Valencia’ sweet orange trees by comparing morphological, transcriptome, and phytohormone profiles at different leaf development phases (1. buds at the start of the experiment; 2. buds on day 5; . 3. leaf emergence; 4. leaf expansion; and 5. leaf maturation) to healthy trees. Over a period of 3 months (in greenhouse conditions), HLB-affected trees had ≈40% reduction in growth traits such as tree height, number of shoots per tree, shoot length, internode length, and leaf size compared to healthy trees. In addition, buds from HLB-affected trees lagged by ≈1 week in sprouting as well as leaf growth. Throughout the leaf development, high accumulation of defense hormones, salicylic acid (SA) and abscisic acid (ABA), and low levels of growth-promoting hormone (auxin) were found in HLB-affected trees compared to healthy trees. Concomitantly, HLB-affected trees had upregulated differentially expressed genes (DEGs) encoding SA, ABA, and ethylene-related proteins in comparison to healthy trees. The total number of cells per leaf was lower in HLB-affected trees compared to healthy trees, which suggests that reduced cell division may coincide with low levels of growth-promoting hormones leading to small leaf size. Both bud dieback and leaf drop were higher in HLB-affected trees than in healthy trees, with concomitant upregulated DEGs encoding senescence-related proteins in HLB-affected trees that possibly resulted in accelerated aging and cell death. Taken together, it can be concluded that HLB-affected trees had a higher tradeoff of resources on defense over growth, leading to sparse canopies and a high tree mortality rate with HLB progression.

The use of canopy density for crop production is a useful tool for evaluating management practices for informed decision-making and predicting crop yields. Traditional methods for analyzing canopy density include expensive equipment that requires advanced software and costly repairs, such as leaf area index analyzers, or equipment that can only be used during optimal weather conditions, such as light meters, that quantify photosynthetically active radiation (PAR), thus making it difficult for researchers to maximize the time needed for field research evaluations and data collection. Digital image analyses using technologically advanced cameras, such as smartphone cameras, have allowed new ways of collecting data without the need to purchase specialized instruments. Using a combination of smartphone cameras and ImageJ software, canopy density can be measured in any weather conditions for a much lower cost than that of traditional equipment. This low-cost, digital image analysis method was compared with traditional PAR measurements for ‘Valencia’ sweet orange [Citrus sinensis (L.) Osb.] trees with varying levels of canopy density. A strong positive correlation between the digital image analysis method and standard canopy density measurement method using PAR measurements (r = 0.79; P

The citrus industry of Florida faces insurmountable challenges against the destructive diseases citrus tristeza and Huanglongbing (HLB, or citrus greening). Though the tristeza causal agent, citrus tristeza virus (CTV), has been in Florida decades longer than HLB, growers have concentrated most of their efforts on combating the more detrimental HLB. The Asian citrus psyllid (Diaphorina citri; ACP) is the insect vector of the bacterial pathogen Candidatus Liberibacter asiaticus and transmits the incurable HLB to all commercial citrus. During our searches for biological and viral controls against the ACP, we consistently detected sequences of CTV in Florida field populations of ACP. This unexpected finding led us to investigate whether ACPs collected from young shoots could be used as a tool to survey CTV in Florida citrus groves. We first surveyed for the most common CTV strains in Florida (T30, T36, and VT/T68) in citrus trees on mostly sour orange (Citrus aurantium) rootstock, the rootstock susceptible to CTV decline. Out of 968 trees sampled across five years (2018-2022), approximately 8.2% were positive for CTV, with more than half of the CTV-positive trees infected with strain T30. Simultaneously, we looked at CTV strains in ACPs during this time and found that approximately 88% of pooled adult and nymph ACPs also had CTV, with over half the positive samples having the T36 strain. As a result of the much higher CTV incidences in the ACPs, we conducted a second investigation into whether we could more easily detect the same CTV strains in ACP nymphs as in CTV-infected citrus tissue. After individually sampling 43 trees and pooling the nymphs from each tree, we detected CTV at about the same incidence in the citrus tissue and the nymphs, but with much less ACP tissue, time, and resources required for detection compared to citrus tissue. Results from this study illustrate the sustained threat of CTV to Florida citrus and demonstrate the ACP as a potential bioindicator for CTV.

This new two-page handout provides a brief summary of citrus leprosis symptoms. Written by Amit Levy, Ozgur Batuman, Megan M. Dewdney, and Jamie D. Burrow and published by the UF/IFAS Plant Pathology Department.

Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom production by activating and modulating plant responses, respectively. The analysis of transcriptome data for CTVΔp33 and the full-length virus suggested that xylem specification has been blocked by detecting several genes encoding xylem, cell wall and lignin degradation, and cell wall loosening enzymes. Furthermore, stem pitting was accompanied by downregulation of transcription factors involved in regulation of xylem differentiation and downregulation of some genes involved in lignin biosynthesis, showing that the xylem differentiation and specification program has been shut off. Upregulation of genes encoding transcription factors associated with phloem and cambium development indicated the activation of this program in stem pitting disease. Furthermore, we detected the induction of several DEGs encoding proteins associated with cell cycle re-entry such as chromatin remodeling factors and cyclin, and histone modification. This kind of expression pattern of genes related to xylem differentiation and specification, phloem and cambium development, and cell cycle re-entry is demonstrated during secondary vascular tissue (SVT) regeneration. The microscopy analysis confirmed that the regeneration of new phloem is associated with stem pitting phenotypes. The findings of this study, thus, provide evidence for the association between stem pitting phenotypes and SVT regeneration, suggesting that the expression of these genes might play important roles in development of stem pitting symptoms. Overall, our findings suggest that phloem regeneration contributes to development of stem pitting symptoms.

The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing (HLB; Citrus greening). This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible ‘Valencia’ sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, β-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected ‘Valencia’ sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and ‘Valencia’ suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ ‘Valencia’ sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.

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The putative causal agent of citrus greening Candidatus Liberibacter asiaticus (CLas) cannot be cultured, which hampers finding new therapies to control this devastating disease. Here, the authors show that hairy roots support CLas propagation and enable high throughput antimicrobial screening.

In this article, we will show evidence that HLB infected trees with a healthy canopy produce higher fruit yields. Surprisingly, we didn’t find that to be true for trees with higher Ct values. Prime attention should be paid to keeping canopy healthy, rather than reducing bacteria titers

Stem pitting disease caused mostly by VT isolates of citrus tristeza virus (CTV) can be a very serious disease on citrus types and varieties that are widely grown in Florida, such as sweet orange and grapefruit. Once a tree is infected with CTV stem pitting, the long-term economic effects to the grower may be even worse than those from decline isolates of CTV. VT isolates that induce mild to moderate stem pitting in grapefruit and sweet orange were previously reported from Florida and are still present here today. Fortunately, CTV isolates causing severe stem pitting have not been detected in Florida yet. It is important to continue to studying CTV and its impact in Florida in order to keep severe stem pitting out of the state. Peer reviewed through UF/IFAS Citrus Research and Education Center; published in Citrus Industry Magazine.

This document is one in a series designed to provide important information on the causal agent, symptoms, and transmission of exotic citrus diseases. This information can be used as an educational tool to raise awareness about these diseases and for scouting and identification efforts. Disseminating information about the diseases to the citrus industry may prevent their introduction and spread in Florida. This document will focus on the exotic viral disease caused by isolates of citrus tristeza virus–stem pitting (CTV-SP). Original version: Chung, Kuang-Ren, and Ronald Brlansky. 2006. “Citrus Diseases Exotic to Florida: Citrus Tristeza Virus– Stem Pitting (CTV-SP)”. EDIS 2006 (7). https://doi.org/10.32473/edis-pp149-2006.

Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

In Florida, almost all citrus trees are affected with Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). We characterized various parameters of HLB-affected sweet orange trees in response to yield-improving nutritional treatment, including canopy volume, canopy density and CLas Ct values, and found that the treatment improved yield and maintained canopy density for over three years, whereas untreated HLB-affected trees declined in canopy density. The nutritional treatment did not affect CLas titer or the tree canopy volume suggesting that canopy density is a better indicator of fruit yield. To further validate the importance of canopy density, we evaluated three independent orchards (different in tree age or variety) to identify the specific traits that are correlated with fruit yields. We found that canopy density and fruit detachment force (FDF), were positively correlated with fruit yields in independent trials. Canopy density accurately distinguished between mild and severe trees in three field trials. High and low producing HLB trees had the same Ct values. Ct values did not always agree with CLas number in the phloem, as visualized by transmission electron microscopy. Our work identifies canopy density as an efficient trait to predict yields of HLB-affected trees and suggests canopy health is more relevant for yields than the CLas population.

Reviewed March 2021. There are no changes in recommendation from 2020.

This article is one in a series designed to provide important information on the causal agent, symptoms, and transmission of exotic citrus diseases. Disseminating the information about the diseases to the citrus industry may prevent their introduction and spread in Florida. This 5-page document will focus on the exotic viral disease citrus leprosis. This is a major revision of an article originally published in 2006. Written by O. Batuman, A. Levy, P. Sieburth, A. M. Paolillo, K.-R. Chung, and R. H. Brlansky, and published by the UF/IFAS Plant Pathology Department. https://edis.ifas.ufl.edu/pp148 Previous version: Chung, Kuang-Ren, and Ronald Brlansky. 2006. “Citrus Diseases Exotic to Florida: Citrus Leprosis”. EDIS 2006 (7). https://journals.flvc.org/edis/article/view/115752.

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To infect their hosts and cause disease, plant viruses must replicate within cells and move throughout the plant both locally and systemically. RNA virus replication occurs on the surface of various cellular membranes, whose shape and composition become extensively modified in the process. Membrane contact sites (MCS) can mediate non-vesicular lipid-shuttling between different membranes and viruses co-opt components of these structures to make their membrane environment suitable for replication. Whereas animal viruses exit and enter cells when moving throughout their host, the rigid wall of plant cells obstructs this pathway and plant viruses therefore move between cells symplastically through plasmodesmata (PD). PD are membranous channels connecting nearly all plant cells and are now viewed to constitute a specialized type of endoplasmic reticulum (ER)-plasma membrane (PM) MCS themselves. Thus, both replication and movement of plant viruses rely on MCS. However, recent work also suggests that for some viruses, replication and movement are closely coupled at ER-PM MCS at the entrances of PD. Movement-coupled replication at PD may be distinct from the main bulk of replication and virus accumulation, which produces progeny virions for plant-to-plant transmission. Thus, MCS play a central role in plant virus infections, and may provide a link between two essential steps in the viral life cycle, replication and movement. Here, we provide an overview of plant virus-MCS interactions identified to date, and place these in the context of the connection between viral replication and cell-to-cell movement.

The destructive citrus disease, Huanglongbing (HLB) or citrus greening, continues to devastate Florida’s citrus industry. A hemipteran insect, the Asian citrus psyllid (ACP), disperses Candidatus Liberibacter asiaticus, one of the putative bacterial pathogens of HLB. This study builds upon ongoing research utilizing high-throughput sequencing to analyze the virome of ACP populations collected from citrus groves throughout Florida. Following the widespread detection of sequences aligning to the genome of citrus tristeza virus (CTV) across consecutive years in the Florida ACP virome, we continued to detect a pervasive amount of CTV in Florida ACPs during subsequent years. Simultaneously, we also detected mixed infections of CTV strains in pooled ACPs from different Florida regions. Predating the HLB epidemic, CTV has been present in Florida for many years and our results confirm its widespread and diverse persistence in Florida citrus groves through a unique lens, the ACP. CTV presence in the ACP likely results from feeding on CTV-infected citrus trees in Florida citrus groves, which may help to understand an overlapping presence of CTV and HLB, both endemic citrus pathosystems in the state, and their role in future integrated pest management strategies.

Callose is a polysaccharide that can be fluorescently stained to study many developmental and immune functions in plants. High-throughput methods to accurately gather quantitative measurements of callose from confocal images are useful for many applications in plant biology. Previous callose quantification methods relied upon binary local thresholding, which had the disadvantage of not being able to differentiate callose in conditions with low contrast from background material. Here, a measurement approach that utilizes the Ilastik supervised machine learning imagery data collection software is described. The Ilastik software method provided superior efficiency for acquiring counts of callose deposits. We also determined the accuracy of these methods as compared to manual counts. We demonstrate that the automated software methods are both good predictors of manual counts, but that the Ilastik counts are significantly closer. Researchers can use this information to guide their choice of method to quantify callose in their work.

Abstract Background Transient gene expression is a powerful tool to study gene function in plants. In citrus, Agrobacterium transformation is the method of choice for transient expression studies, but this method does not work efficiently with many gene constructs, and there is a need for a more robust transient expression system in citrus leaves. Biolistic particle delivery is an alternative to Agrobacterium transformation, and in some plants, such as Arabidopsis, gives higher transformation rates in leaf tissues than Agrobacterium. Results Here we describe an improved method for gene expression in epidermal cells of citrus leaves, using the Bio-Rad Helios gene-gun. Gene-gun bombardment of GFP-HDEL produced highly efficient gene expression in large number of cells and in different citrus varieties. We show here that transiently expressed proteins have maintained their functions in plants, and this is demonstrated by the subcellular localization of different organelle markers, and by a functional assay of Xanthomonas citri effector AvrGF1. To further expand the available tools for subcellular localization studies in citrus, we also generated a new set of transgenic citrus plants that contain organelle markers labelling the nuclei, actin and endoplasmic reticulum. Using these new tools, we were able to show that the coat protein of citrus tristeza virus localizes to the cytoplasm and nuclei when expressed in epidermal cells fused to GFP. Conclusion We have optimized a new method for transient expression in citrus leaves, to give highly reproducible and efficient transformation without producing a high level of injury or artifacts to the bombarded tissue. We also generated the first set organelle markers for use in citrus. These fluorescent protein markers label the nucleus and the actin. With these new resources, protein activity and subcellular localization can be studied in citrus rapidly and in high throughput. The handheld gene-gun device can also be used in the grove to deliver therapies for citrus diseases, such as canker and Huanglongbing, into trees.

The plant pathogenic bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of the citrus disease Huanglongbing (HLB), and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri), have been devastating the Florida citrus industry. To restore the competitive production presence of Florida in the worldwide citrus market, effective and sustainable control of HLB and the ACP needs to be identified. As alternatives for resistance-inducing insecticides, viruses are currently being considered for biological control of the ACP. To identify possible biological control candidates, we conducted one of the most comprehensive surveys of natural ACP populations in major citrus production regions spanning 21 counties in Florida. By optimizing PCRs and RT-PCRs, we were able to successfully detect and monitor the prevalence of five previously identified ACP-associated RNA and DNA viruses throughout Florida citrus groves, which include: Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV), Diaphorina citri densovirus (DcDNV), Diaphorina citri reovirus (DcRV), and Diaphorina citri picorna-like virus (DcPLV). Adult and nymph ACP populations from 21 of Florida's major citrus-producing counties were collected each month during approximately 18 consecutive months. RNA extracts used for these viral screens were also regionally combined and subjected to High Throughput Sequencing (HTS) to reveal a more comprehensive picture of known and unknown viruses in Florida ACP populations. We discovered that DcACV was the most prevalent ACP-associated virus throughout nymph and adult ACP populations in Florida, detected in more than 60% of all samples tested, followed by DcPLV and DcFLV. HTS allowed us to identify a novel ACP-associated reo-like virus and a picorna-like virus. The putative reo-like virus, tentatively named Diaphorina citri cimodo-like virus, was later surveyed and detected back in seasonal adult and nymph ACP samples collected in Florida during this study. HTS generated data also revealed that the most abundant virus in Florida ACP populations was Citrus tristeza virus (CTV), which is not an ACP-associated virus, suggesting persistent presence of CTV infection in citrus throughout Florida groves. Collectively, information obtained from our study may be able to help guide the direction of biotechnological pest control efforts involving a number of viruses that were detected for the first time in Florida ACP populations, including two newly identified ACP-associated viruses.

As a practical alternative to chemical controls, development of knowledge-based and efficient biological control strategies to disrupt HLB spread by current technologies (i.e., RNAi) in the ACP vector itself represent our strategy to control the disease without relying solely on chemical applications. The necessity of a precise and specific biological control for Florida ACP populations initiated our ongoing study, with hopes to provide additional molecular tools. In order to decrease or eliminate major dependence on broad-spectrum chemical controls, our study aims to investigate the Florida ACP body (i.e., gut) for identifying an ACP-associated virus with the potential to be manipulated into a biocontrol agent against the HLB insect vector. The endemic establishment of both HLB and the ACP in Florida has greatly increased the need for alternative control options.

Citrus tristeza virus (CTV) is a major cause of the decline and eventual death of trees on sour orange rootstocks. This 2-page fact sheet is part of the 2019–2020 Florida Citrus Production Guide. Written by O. Batuman, A. Levy, M. E. Hilf, P. J. Sieburth, W. O. Dawson, and R. H. Brlansky, and published by the Plant Pathology Department, March 2019. PP-181/CG039: 2022–2023 Florida Citrus Production Guide: Tristeza Decline (ufl.edu)

Exocortis and cachexia are diseases caused by viroids, which can lead to stunted growth and reduced yields in affected plants. Viroids are small, infectious circular-RNA molecules. Exocortis causes dwarfing and bark scaling on rootstocks such as trifoliate orange and many of its hybrids such as Carrizo citrange, Rangpur lime, and others. This 2-page fact sheet is part of the 2019–2020 Florida Citrus Production Guide. Written by Ozgur Batuman, Amit Levy, and Ronald H. Brlansky, and published by the Plant Pathology Department, March 2019. PP-179/CG037: 2022–2023 Florida Citrus Production Guide: Exocortis, Cachexia, and Other Viroids (ufl.edu)

Exocortis and cachexia are diseases caused by viroids, which can lead to stunted growth and reduced yields in affected plants. Viroids are small, low-molecular-weight, infectious RNA molecules. This 1-page fact sheet is part of the 2018–2019 Florida Citrus Production Guide. Written by Ozgur Batuman, Amit Levy, and Ronald H. Brlansky, and published by the UF/IFAS Plant Pathology Department, May 2018. PP-179/CG037: 2022–2023 Florida Citrus Production Guide: Exocortis, Cachexia, and Other Viroids (ufl.edu)

When thinking about genetic engineering, the first thing that comes to mind is usually GMOs (genetically modified organisms) In a GMO, a plant genome is engineered to include additional genetic sequences, usually genes with some useful activity that give the plant an advantage. This method is called a stable method because genes will be inherited to the next generation. It is usually very slow, especially with tree crops such as citrus, which have a long juvenile stage that ranges from four to six years. It makes introducing and evaluating any genetic trait a two-decade process. However, this is only one possibility.

Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria−host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

β-1,3-Glucanases (BG) have been implicated in enhancing virus spread by degrading callose at plasmodesmata (Pd). Here, we investigate the role of Arabidopsis BG in tobamovirus spread. During Turnip vein clearing virus infection, the transcription of two pathogenesis-related (PR)-BG AtBG2 and AtBG3 increased but that of Pd-associated BG AtBG_pap did not change. In transgenic plants, AtBG2 was retained in the endoplasmic reticulum (ER) network and was not secreted. As a stress response mediated by salicylic acid, AtBG2 was secreted and appeared as a free extracellular protein localized in the entire apoplast but did not accumulate at Pd sites. At the leading edge of Tobacco mosaic virus spread, AtBG2 co-localized with the viral movement protein in the ER-derived bodies, similarly to other ER proteins, but was not secreted to the cell wall. In atbg2 mutants, callose levels at Pd and virus spread were unaffected. Likewise, AtBG2 overexpression had no effect on virus spread. However, in atbg_pap mutants, callose at Pd was increased and virus spread was reduced. Our results demonstrate that the constitutive Pd-associated BG but not the stress-regulated extracellular PR-BG are directly involved in regulation of callose at Pd and cell-to-cell transport in Arabidopsis, including the spread of viruses.

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.