TGB1 and TGB2 also localized with membrane vesicles even though extensive membrane proliferation was not observed. However, ectopically expressed TGB3 elicited formation of a complex and well-defined ER network that is closely associated with, or houses, thick actin cables and TGB proteins.Virus movement and membrane reorganization, which is especially obvious in the perinuclear region, was disrupted by mutations in the central membranespanning domain of TGB3. Our findings thus suggest that host membrane associations are involved in several aspects of BSMV movement that merit future study. In addition to differences in requirements of the coat protein for cell-to-cell movement of hordeiviruses and potexviruses, a number of variations are evident in TGB1 protein structure, biochemical activities, interference with host gene silencing, and movement functions of the virgaviruses and the TGB-containing flexiviruses . A recent paper illuminating a TGB1 requirement for formation of X bodies associated with PVX highlights another major difference in functions of the TGB1 proteins of the hordeiviruses and the potexviruses that relates to BSMV actin remodeling. In contrast to BSMV, in which TGB3 expression has a major effect on actin architecture, PVX TGB1 is essential for X-body formation and functions in extensive actin and membrane remodeling . The multilayered membranous X-body is an important organelle that is required for normal levels of viral RNA replication and virion accumulation. Nevertheless, in plants and protoplasts infected with PVX mutants unable to express TGB1, morphogenesis of X bodies fails to occur, yet low levels of PVX replication can be detected and small amounts of virus particles accumulate . However, ectopic expression of PVX TGB1 results in massive remodeling of host actin and endomembranes, greenhouse ABS snap clamp and recruitment of these structures, as well as TGB2 and TGB3, to sites near the nucleus. Subsequently, X-bodies develop into complex multilayered membrane organelles adjacent to the nucleus, that selectively incorporate TGB proteins, ribosomes, viral RNA and virions to specific sites within the granular vesicular bodies .
These differences between the two viruses are further illustrated by conventional electron microscopic observations showing that structures corresponding to PVX X-bodies are not present in BSMV-infected cells, and that BSMV replicates in membrane vesicles formed from the chloroplast outer membrane . Our current results add to a growing list of major differences in the movement processes of TGB-encoding viruses . We previously reported that cytochalasin D treatment failed to affect TGB1 localization in BSMV infected protoplasts and as a result, postulated that cytoskeletal interactions of the protein were relatively minor. However, the experiments presented here reveal both actin remodeling and changes to ER structure as a consequence of BSMV infection and transient expression of TGB3 and TGB2/3. Our observations also provide evidence that the subcellular localization of the TGB proteins depends on actin cytoskeleton interactions. To investigate these interactions in more detail, we used LatB to inhibit actin polymerization in cells infiltrated with TGBreporter proteins. In contrast to cytochalasin D used in our earlier experiments , LatB can be up to 100-fold more potent than cytochalasins, and functions by shortening and thickening of actin filaments. After LatB treatment, the DsRed:Talin patterns in N. benthamiana infiltrated epidermal leaf cells exhibited a major shift from a filamentous actin network to thick cablelike structures.From these experiments, we conclude that actin cytoskeleton modifications are required for BSMV movement and that TGB3 has a critical role in cytoskeleton remodeling during movement. In contrast to the BSMV LatB experiments described above, experiments with the closely related PSLV TGB3 have resulted in different conclusions about mechanisms functioning in PD targeting . In the case of BSMV, actin cytoskeleton disruption by LatB interfered with CW localization of TGB3, and TGB1 when coexpressed with TGB2/3, whereas PSLV TGB3 CW localization was not dramatically affected by LatB treatment . These disparate results highlight fundamental differences in the mechanisms of subcellular transit of BSMV and PSLV. Such differences between related viruses may occur more often than previously realized, as illustrated by a previous report describing differences in the movement of two tobamoviruses .
In this direct comparison, movement of TMV is strongly inhibited by LatB treatment, whereas movement of the related TVCV is unaffected by LatB treatment. These results argue strongly that more than one mechanism may be operative in some closely related viruses, and our collective results suggest that BSMV and PSLV may fit within this category. Evidence for TGB3 associations with the Golgi membranes during coexpression of DsRed:TGB3 and the STGFP Golgi marker indicates that Golgi derived vesicles and DsRed:TGB3 co-localize with the CW after plasmolysis. BFA interference with Golgi stack integrity resulted in a major collapse of vesicles localized in close proximity to the CW, but BFA appears to have only limited effects on BSMV localization or PSLV TGB3 associations with “peripheral bodies” . Nevertheless, differences in the BSMV and PSLV LatB cytoskeleton disruption experiments suggest that different mechanisms may function in some TGB3 interactions culminating in PD targeting. Other than the preliminary experiments shown above, which suggest that BSMV infection does not result in obvious changes to microtubules, we have not extensively investigated possible direct interactions of BSMV TGB proteins with microtubules. However, in other experiments with the related Potato mop-top virus , colchicine treatments were used to disrupt tubulin polymerization and microtubule integrity . Colchicine can affect multiple metabolic and regulatory processes affecting a large number of functions that might interfere with TGB1 localization to the CW. However, the PD associations of PMTV mutants provided evidence for an association between microtubules and PMTV TGB1. Of particular interest, cells were observed for several days after transient expression of the three TGB proteins in ratios corresponding to those occurring during virus infection. During this period, a defined series of kinetic events were noted, beginning with PMTV TGB1 nucleolar interactions and proceeding through cytoplasmic granules to the CW. Thus, the effects of BSMV and PMTV on microtubule remodeling, seem to differ, and these experiments reinforce our suggestion that multiple pathways may operate in CW targeting during TGB1 expression of the virgaviruses. Unfortunately, individual events involved in viral movement from subcellular sites of replication to the PD and adjacent cells are difficult to dissect experimentally, and many of these problems have been discussed previously .
The infection front where important events are coordinated is a moving boundary consisting of a limited number of cells undergoing a series of asynchronous steps, so relatively few studies have probed events at this stage of infection. Variations in delivery protocols also contribute to experimental differences or artifacts that can lead to aberrant subcellular trafficking effects. In this regard,flower pot wholesale examples of the effects of over expression of PSLV TGB3 has been described recently in which anomalous cell death, membrane abnormalities and disrupted Golgi functions occur during transient infection . Third, pharmacological approaches can be quite variable in the hands of different researchers. Finally, a more diverse array of approaches, including infectivity studies applied to different hosts might provide interesting insights into alternative strategies employed by BSMV and other hordeiviruses. Although it would be preferable to investigate movement in the natural BSMV cereal hosts, these plants present technical difficulties that are difficult to circumvent. Fortunately, BSMV, unlike PSLV, is able to infect N. benthamiana, so we have been able to compare cytological and biochemical experiments with infectivity results in this host.Rust fungi are an order of >7000 species of highly specialized plant pathogens with a disproportionately large impact on agriculture, horticulture, forestry, and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. Asian soybean rust caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is a prime example of the damage that can be caused by rust fungi. It is a critical challenge for food security and one of the most damaging plant pathogens of this century. The disease is ubiquitously present in the soybean growing areas of Latin America, where 210 million metric tons of soybean are projected to be produced in 2022/23 , and on average representing a gross production value of U.S. $ 115 billion per season . A low incidence of this devastating disease can already affect yields and, if not managed properly, yield losses are reported of up to 80%. Chemical control in Brazil to manage the disease started in the 2002/03 growing season. In the following season, ~20 million hectares of soybeans were sprayed with fungicides to control this disease. The cost of managing P. pachyrhizi exceeds $2 billion USD per season in Brazil alone. The pathogen is highly adaptive and individually deployed resistance genes have been rapidly overcome when respective cultivars have been released. Similarly, the fungal tolerance to the main classes of site-specific fungicides is increasing, making chemical control less effective. Another remarkable feature for an obligate biotrophic pathogen is its wide host range, encompassing 153 species of legumes within 54 genera to date. Epidemiologically, this is relevant as it allows the pathogen to maintain itself in the absence of soybean on other legume hosts, such as overwintering on the invasive weed Kudzu in the United States. Despite the importance of the pathogen, not much was known about its genetic makeup as the large genome size , coupled to a high repeat content, high levels of heterozygosity and the dikaryotic nature of the infectious urediospores of the fungus have hampered whole genome assembly efforts. In this work, we provide reference quality assemblies and genome annotations of three P. pachyrhizi isolates. We uncover a genome with a total assembly size of up to 1.25 Gb.
Approximately, 93% of the genome consists of TEs, of which two super families make up 80% of the TE content. The three P. pachyrhizi isolates collected from South America represent a single clonal lineage with high levels of heterozygosity. Studying the TEs in detail, we demonstrate that the expansion of TEs within the genome happened over the last 10 My and accelerated over the last 3 My, and did so in several bursts. Although TEs are tightly controlled during sporulation and appressoriaformation, we can see a clear relaxation of repression during the in planta life stages of the pathogen. Due to the nested TEs, it is not possible at present to correlate specific TEs to specific expanded gene families. However, we can see that the P. pachyrhizi genome is expanded in genes related to amino acid metabolism and energy production, which may represent key lifestyle adaptations. Overall, our data unveil that TEs that started their proliferation during the radiation of the Leguminosae play a prominent role in the P. pachyrhizi’s genome and may have a key impact on a variety of processes such as host range adaptation, stress responses and plasticity of the genome. The high-quality genome assembly and transcriptome data presented here are a key resource for the community. It represents a critical step for further in-depth studies of this pathogen to develop new methods of control and to better understand the molecular dialogue between P. pachyrhizi and its agriculturally relevant host, Soybean.The high repeat content and dikaryotic nature of the P. pachryrhizi genome poses challenges to genome assembly methods. Recent improvements in sequencing technology and assembly methods have provided contiguous genome assemblies for several rust fungi. Here, we have expanded the effort and provided reference-levelgenome assemblies of three P. pachyrhizi isolates using long-read sequencing technologies. All three isolates were collected from different regions of South America. We have used PacBio sequencing for the K8108 and MT2006 isolates and Oxford Nanopore for the UFV02 isolate to generate three high-quality genomes . Due to longer read lengths from Oxford nanopore, the UFV02 assembly is more contiguous compared to K8108 and MT2006 and is used as a reference in the current study . The total genome assembly size of up to 1.25 Gb comprising two haplotypes, makes the P. pachyrhizi genome one of the largest fungal genomes sequenced to date . Analysis of the TE content in the P. pachyrhizi genome indicates ~93% of the genome consist of repetitive elements, one of the highest TE contents reported for any organism to date . This high TE content may represent a key strategy to increase genetic variation in P. pachyrhizi. The largest class of TEs are class 1 retrotransposons, that account for 54.0% of the genome.