Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Atypical molecular features of RNA silencing against the phloem-restricted polerovirus TuYV.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.

Exosomes mediate horizontal transmission of viral pathogens from insect vectors to plant phloem.

Numerous piercing-sucking insects can horizontally transmit viral pathogens together with saliva to plant phloem, but the mechanism remains elusive. Here, we report that an important rice reovirus has hijacked small vesicles, referred to as exosomes, to traverse the apical plasmalemma into saliva-stored cavities in the salivary glands of leafhopper vectors. Thus, virions were horizontally transmitted with exosomes into rice phloem to establish the initial plant infection during vector feeding. The purified exosomes secreted from cultured leafhopper cells were enriched with virions. Silencing the exosomal secretion-related small GTPase Rab27a or treatment with the exosomal biogenesis inhibitor GW4869 strongly prevented viral exosomal release in vivo and in vitro. Furthermore, the specific interaction of the 15-nm-long domain of the viral outer capsid protein with Rab5 induced the packaging of virions in exosomes, ultimately activating the Rab27a-dependent exosomal release pathway. We thus anticipate that exosome-mediated viral horizontal transmission is the conserved strategy hijacked by vector-borne viruses.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Semipersistently Transmitted, Phloem Limited Plant Viruses Are Inoculated during the First Subphase of Intracellular Stylet Penetrations in Phloem Cells.

The green peach aphid Myzus persicae Sulzer is the main vector of the semipersistently transmitted and phloem-limited Beet yellows virus (BYV, Closterovirus). Studies monitoring the M. persicae probing behavior by using the Electrical penetration graphs (EPG) technique revealed that inoculation of BYV occurs during unique brief intracellular punctures (phloem-pds) produced in companion and/or sieve element cells. Intracellular stylet punctures (or pds) are subdivided in three subphases (II-1, II-2 and II-3), which have been related to the delivery or uptake of non-phloem limited viruses transmitted in a non-persistent or semipersistent manner. As opposed to non-phloem limited viruses, the specific pd subphase(s) involved in the successful delivery of phloem limited viruses by aphids remain unknown. Therefore, we monitored the feeding process of BYV-carrying M. persicae individuals in sugar beet plants by the EPG technique and the feeding process was artificially terminated at each phloem-pd subphase. Results revealed that aphids that only performed the subphase II-1 of the phloem-pd transmitted BYV at similar efficiency than those allowed to perform subphase II-2 or the complete phloem-pd. This result suggests that BYV inoculation occurs during the first subphase of the phloem-pd. The specific transmission mechanisms involved in BYV delivery in phloem cells are discussed.

Dynamic changes impact the plum pox virus population structure during leaf and bud development.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host.

Viral Hacks of the Plant Vasculature: The Role of Phloem Alterations in Systemic Virus Infection.

For plant viruses, the ability to load into the vascular phloem and spread systemically within a host is an essential step in establishing a successful infection. However, access to the vascular phloem is highly regulated, representing a significant obstacle to virus loading, movement, and subsequent unloading into distal uninfected tissues. Recent studies indicate that during virus infection, phloem tissues are a source of significant transcriptional and translational alterations, with the number of virus-induced differentially expressed genes being four- to sixfold greater in phloem tissues than in surrounding nonphloem tissues. In addition, viruses target phloem-specific components as a means to promote their own systemic movement and disrupt host defense processes. Combined, these studies provide evidence that the vascular phloem plays a significant role in the mediation and control of host responses during infection and as such is a site of considerable modulation by the infecting virus. This review outlines the phloem responses and directed reprograming mechanisms that viruses employ to promote their movement through the vasculature.

Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana.

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

Impact of Mutations in Arabidopsis thaliana Metabolic Pathways on Polerovirus Accumulation, Aphid Performance, and Feeding Behavior.

During the process of virus acquisition by aphids, plants respond to both the virus and the aphids by mobilizing different metabolic pathways. It is conceivable that the plant metabolic responses to both aggressors may be conducive to virus acquisition. To address this question, we analyze the accumulation of the phloem-limited polerovirus Turnip yellows virus (TuYV), which is strictly transmitted by aphids, and aphid's life traits in six Arabidopsis thaliana mutants (xth33, ss3-2, nata1, myc234, quad, atr1D, and pad4-1). We observed that mutations affecting the carbohydrate metabolism, the synthesis of a non-protein amino acid and the glucosinolate pathway had an effect on TuYV accumulation. However, the virus titer did not correlate with the virus transmission efficiency. Some mutations in A.thaliana affect the aphid feeding behavior but often only in infected plants. The duration of the phloem sap ingestion phase, together with the time preceding the first sap ingestion, affect the virus transmission rate more than the virus titer did. Our results also show that the aphids reared on infected mutant plants had a reduced biomass regardless of the mutation and the duration of the sap ingestion phase.

Defenses against Virus and Vector: A Phloem-Biological Perspective on RTM- and SLI1-Mediated Resistance to Potyviruses and Aphids.

Combining plant resistance against virus and vector presents an attractive approach to reduce virus transmission and virus proliferation in crops. RestrictedTobacco-etch virus Movement (RTM) genes confer resistance to potyviruses by limiting their long-distance transport. Recently, a close homologue of one of the RTM genes, SLI1, has been discovered but this gene instead confers resistance to Myzus persicae aphids, a vector of potyviruses. The functional connection between resistance to potyviruses and aphids, raises the question whether plants have a basic defense system in the phloem against biotic intruders. This paper provides an overview on restricted potyvirus phloem transport and restricted aphid phloem feeding and their possible interplay, followed by a discussion on various ways in which viruses and aphids gain access to the phloem sap. From a phloem-biological perspective, hypotheses are proposed on the underlying mechanisms of RTM- and SLI1-mediated resistance, and their possible efficacy to defend against systemic viruses and phloem-feeding vectors.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.

Barley yellow dwarf virus Can Be Inoculated During Brief Intracellular Punctures in Phloem Cells Before the Sieve Element Continuous Salivation Phase.

The distinguished intracellular stylet puncture called phloem-pd (potential drop [pd]) produced by Myzus persicae has been associated with the transmission of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus). However, the production of intracellular punctures in phloem cells (phloem-pd) by other aphid species and their role in the transmission of persistently transmitted, phloem-limited viruses are still unknown. Previous studies revealed that inoculation of the persistently transmitted, phloem-limited Barley yellow dwarf virus (BYDV, Luteovirus) is associated mainly with the sieve element continuous salivation phase (E1 waveform). However, the role of brief intracellular punctures that occur before the E1 phase in the inoculation of BYDV by aphids is unknown. We aimed to investigate whether the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae) produced a stereotypical phloem-pd and to study its role in the inoculation of BYDV. The feeding behavior of viruliferous R. padi individuals in barley (Hordeum vulgare) was monitored via the electrical penetration graph (EPG) technique. The feeding process was artificially terminated after the observation of specific EPG waveforms: standard-pds, phloem-pd, and E1. Analysis of the EPG recordings revealed the production of a phloem-pd pattern by R. padi, in addition to a short, distinct E1-like pattern (short-E1), both resulting in successful inoculation of BYDV. Also, the transmission efficiency of BYDV was directly proportional to the time spent by aphids in intracellular salivation in phloem cells. Finally, we discussed the main differences between the inoculation process of semipersistent and persistently transmitted phloem-limited viruses by aphids.

Combining Transient Expression and Cryo-EM to Obtain High-Resolution Structures of Luteovirid Particles.

The Luteoviridae are pathogenic plant viruses responsible for significant crop losses worldwide. They infect a wide range of food crops, including cereals, legumes, cucurbits, sugar beet, sugarcane, and potato and, as such, are a major threat to global food security. Viral replication is strictly limited to the plant vasculature, and this phloem limitation, coupled with the need for aphid transmission of virus particles, has made it difficult to generate virus in the quantities needed for high-resolution structural studies. Here, we exploit recent advances in heterologous expression in plants to produce sufficient quantities of virus-like particles for structural studies. We have determined their structures to high resolution by cryoelectron microscopy, providing the molecular-level insight required to rationally interrogate luteovirid capsid formation and aphid transmission, thereby providing a platform for the development of preventive agrochemicals for this important family of plant viruses.

A Distinct, Non-Virion Plant Virus Movement Protein Encoded by a Crinivirus Essential for Systemic Infection.

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamianaIMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

Newly Distinguished Cell Punctures Associated with Transmission of the Semipersistent Phloem-Limited Beet Yellows Virus.

Here we report on plant penetration activities (probing) by the aphid Myzus persicae (Sulzer, 1776) in association with the transmission, acquisition, and inoculation of the semipersistent Beet yellows virus (BYV; Closterovirus) in sugar beet. During electrical penetration graph (EPG) recording of stylet pathways, standard intracellular stylet punctures occur which are called potential drop (pd) waveforms. In addition to the standard pd, there also appeared to be a unique type of intracellular stylet puncture that always preceded the phloem salivation phase (waveform E1). This type of pd, the phloem-pd, showed properties distinct from those of the standard pds and has never been described before. We manually ended EPG recordings during the acquisition and inoculation tests by removing aphids from the source or test plant after specific waveforms were recorded. Inoculation of BYV occurred at the highest rate when probing was interrupted just after a single or various phloem-pds. In contrast, BYV acquisition showed an intimate association with sustained phloem sap ingestion from phloem sieve elements (SEs) (E2 waveform). Our work shows for the first time that the inoculation of a phloem-limited virus occurs during specific intracellular stylet punctures and before phloem salivation (waveform E1). Further studies are needed to establish in what cells this novel phloem-pd occurs: phloem parenchyma, companion, or SE cells. The role of the different stylet activities in the acquisition and inoculation of BYV by M. persicae is discussed.IMPORTANCE We discovered the specific feeding activities of Myzus persicae (Sulzer, 1776) associated with the transmission of Beet yellows virus (BYV; Closterovirus). Our work strongly suggests that aphids can insert their stylets into the membranes of phloem cells-visualized as a unique type of waveform that is associated with the inoculation of BYV. This intracellular puncture (3 to 5 s) occurs just before the phloem salivation phase and can be distinguished from other nonvascular stylet cell punctures. This is the first time that the transmission of a phloem-limited semipersistent virus has been shown to be associated with a unique type of intracellular puncture. Our work offers novel information and strongly contributes to the existing literature on the transmission of plant viruses. Here we describe a new kind of aphid behavioral pattern that could be key in further works, such as studying the transmission of other phloem-limited viruses (e.g., luteoviruses).

Development of a GFP expression vector for Cucurbit chlorotic yellows virus.

BACKGROUND: Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date. FINDING: We constructed a CCYV GFP expression vector using the "add a gene" strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFPSGC, pCCYVGFPCGC, and pCCYVGFPCGS, were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFPCGC-infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFPSGC and pCCYVGFPCGS were mainly found in single cells. Further observation of pCCYVGFPCGC GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves. CONCLUSIONS: We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.

Disruption of Chloroplast Function Through Downregulation of Phytoene Desaturase Enhances the Systemic Accumulation of an Aphid-Borne, Phloem-Restricted Virus.

Chloroplasts play a central role in pathogen defense in plants. However, most studies explaining the relationship between pathogens and chloroplasts have focused on pathogens that infect mesophyll cells. In contrast, the family Luteoviridae includes RNA viruses that replicate and traffic exclusively in the phloem. Recently, our lab has shown that Potato leafroll virus (PLRV), the type species in the genus Polerovirus, forms an extensive interaction network with chloroplast-localized proteins that is partially dependent on the PLRV capsid readthrough domain (RTD). In this study, we used virus-induced gene silencing to disrupt chloroplast function and assess the effects on PLRV accumulation in two host species. Silencing of phytoene desaturase (PDS), a key enzyme in carotenoid, chlorophyll, and gibberellic acid (GA) biosynthesis, resulted in a substantial increase in the systemic accumulation of PLRV. This increased accumulation was attenuated when plants were infected with a viral mutant that does not express the RTD. Application of GA partially suppressed the increase in virus accumulation in PDS-silenced plants, suggesting that GA signaling also plays a role in limiting PLRV infection. In addition, the fecundity of the aphid vector of PLRV was increased when fed on PDS-silenced plants relative to PLRV-infected plants.

Hitchhikers, highway tolls and roadworks: the interactions of plant viruses with the phloem.

The phloem is of central importance to plant viruses, providing the route by which they spread throughout their host. Compared with virus movement in non-vascular tissue, phloem entry, exit, and long-distance translocation usually involve additional viral factors and complex virus-host interactions, probably, because the phloem has evolved additional protection against these molecular 'hitchhikers'. Recent progress in understanding phloem trafficking of endogenous mRNAs along with observations of membranous viral replication 'factories' in sieve elements challenge existing conceptions of virus long-distance transport. At the same time, the central role of the phloem in plant defences against viruses and the sophisticated viral manipulation of this host tissue are beginning to emerge.

The enemy within: phloem-limited pathogens.

The growing impact of phloem-limited pathogens on high-value crops has led to a renewed interest in understanding how they cause disease. Although these pathogens cause substantial crop losses, many are poorly characterized. In this review, we present examples of phloem-limited pathogens that include intracellular bacteria with and without cell walls, and viruses. Phloem-limited pathogens have small genomes and lack many genes required for core metabolic processes, which is, in part, an adaptation to the unique phloem environment. For each pathogen class, we present multiple case studies to highlight aspects of disease caused by phloem-limited pathogens. The pathogens presented include Candidatus Liberibacter asiaticus (citrus greening), Arsenophonus bacteria, Serratia marcescens (cucurbit yellow vine disease), Candidatus Phytoplasma asteris (Aster Yellows Witches' Broom), Spiroplasma kunkelii, Potato leafroll virus and Citrus tristeza virus. We focus on commonalities in the virulence strategies of these pathogens, and aim to stimulate new discussions in the hope that widely applicable disease management strategies can be found.

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants.

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.