Molecular characterization of two new alternaviruses identified in members of the fungal family Nectriaceae.

Since the first report in 2009, at least ten additional viruses have been identified and assigned to the proposed virus family Alternaviridae. Here we report two new mycoviruses tentatively assigned to this family, both identified as members of the fungal family Nectriaceae, which were isolated from surface-disinfected apple roots (Malus x domestica, Borkh.) affected by apple replant disease (ARD). ARD is a highly complex, worldwide-occurring disease resulting from plant reactions to a disturbed (micro)-biome and leads to high economic losses every year. The first alternavirus characterized in this study was identified in a Dactylonectria torresensis isolate. The virus was tentatively named dactylonectria torresensis alternavirus 1 (DtAV1) as the first member of the proposed new species Alternavirus dactylonectriae. The second virus was identified in an isolate of Ilyonectria robusta and was tentatively named ilyonectria robusta alternavirus 1 (IrAV1) as the first member of the proposed new species Alternavirus ilyonectriae. Full genomic sequences of the viruses were determined and are presented. Further, we found hints for putative components of a methyl transferase machinery using in silico approaches. This putative protein domain is encoded by segment 2. However, this result only establishes the basis for subsequent studies in which the function must be confirmed experimentally in vitro. Thus, this is the first study where a function is predicted to all three genomic segments within the group of the alternaviruses. These findings provide further insights into the virome of ARD-associated fungi and are therefore another brick in the wall of understanding the complexity of the disease.

The complete genome sequence of Neckar River virus confirms it to be a distinct member of the genus Tombusvirus in the family Tombusviridae.

Neckar River virus (NRV), first isolated from a water sample of the Neckar River (Germany) in the 1980s, was serologically characterized as a novel tombusvirus. In this study, the complete genome sequence was determined, and an infectious full-length cDNA clone was constructed. The genome organization of NRV (DSMZ PV-0270) resembles that of tombusviruses. The genome consists of 4739 nucleotides and contains five open reading frames (ORFs) and one additional putative ORF (pX) in the 3'-terminal region. Phylogenetic analysis and sequence comparisons confirmed NRV to be a member of the species Tombusvirus neckarfluminis in the genus Tombusvirus. The infectious full-length cDNA clone was constructed using Gibson assembly and subsequent infection of Nicotiana benthamiana plants by Rhizobium radiobacter inoculation. The virus derived from the full-length cDNA clone caused symptoms resembling those caused by the wild-type virus, but slightly milder.

Comparative analysis of virus pathogenicity and resistance-breaking between the P- and A-type from the beet necrotic yellow vein virus using infectious cDNA clones.

The A-type of beet necrotic yellow vein virus (BNYVV) is widely distributed in Europe and is one of the major virus types causing rhizomania disease in sugar beet. The closely related P-type is mainly limited to a small region in France (Pithiviers). Both virus types possess four RNAs (RNA1-4), but the P-type harbours an additional fifth RNA species (RNA5). The P-type is associated with stronger disease symptoms and resistance-breaking of Rz1, one of the two resistance genes which are used to control BNYVV infection. These characteristics are presumably due to the presence of RNA5, but experimental evidence for this is lacking. We generated the first infectious cDNA clone of BNYVV P-type to study its pathogenicity in sugar beet in comparison to a previously developed A-type clone. Using this tool, we confirmed the pathogenicity of the P-type clone in the experimental host Nicotiana benthamiana and two Beta species, B. macrocarpa and B. vulgaris. Independent of RNA5, both the A- and the P-type accumulated in lateral roots and reduced the taproot weight of a susceptible sugar beet genotype to a similar extent. In contrast, only the P-type clone was able to accumulate a virus titre in an Rz1-resistant variety whereas the A-type clone failed to infect this variety. The efficiency of the P-type to overcome Rz1 resistance was strongly associated with the presence of RNA5. Only a double resistant variety, harbouring Rz1 and Rz2, prevented an infection with the P-type. Reassortment experiments between the P- and A-type clones demonstrated that both virus types can exchange whole RNA components without losing the ability to replicate and to move systemically in sugar beet. Although our study highlights the close evolutionary relationship between the two virus types, we were able to demonstrate distinct pathogenicity properties that are attributed to the presence of RNA5 in the P-type.

First report of a chrysovirus infecting a member of the fungal genus Ilyonectria.

The fungus Ilyonectria pseudodestructans belongs to the family Nectriaceae and was found to be part of the endophytic microbiome of apple trees (Malus x domestica, Borkh.) with apple replant disease (ARD). After dsRNA extraction, a mycoviral infection became evident. Here, we report the identification of a new virus, tentatively named "Ilyonectria pseudodestructans chrysovirus 1" (IpCV1), as the first member of the proposed new species "Alphachrysovirus ilyonectriae" within the genus Alphachrysovirus. This is the first report of a chrysovirus infecting a member of the fungal genus Ilyonectria. IpCV1 has a tripartite dsRNA genome with a total length of 8944 bp. The segments are 3439 bp, 2850 bp, and 2655 bp in length, and each dsRNA carries a single ORF. The encoded viral proteins are a 125.92-kDa RNA-dependent RNA polymerase, a 100.75-kDa coat protein, and one protein of unknown function with a predicted molecular mass of 93.04 kDa. The 5´ and 3´ UTRs are comparatively short and are 79 to 91 bp and 62 to 148 bp in length, respectively. This study provides the basis for further investigations of the impact of IpCV1 on its host and the etiology of ARD.

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa.

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.

Complete genome sequence of a German isolate of spartina mottle virus supports its classification as a member of the proposed genus "Sparmovirus" within the family Potyviridae.

Spartina mottle virus (SpMV), an unassigned member of the family Potyviridae, has been known since 1980, when it was first described in England and Wales in symptomatic plants of the genus Spartina. In infected cells, flexuous particles and pinwheel inclusion bodies were found that resemble those of potyvirids. To date, the NCBI database contains only two partial sequences of a German (Nessmersiel) and an Italian (Assisi) isolate, suggesting that SpMV could be the first member of a new genus, called "Sparmovirus", in the family Potyviridae. In this study, the first complete genome sequence of the German SpMV isolate (SpMV Ger) was determined. The genome of SpMV is a single-stranded, monopartite, polyadenylated RNA consisting of 9376 nucleotides. Sequence analysis revealed a genome organization similar to that of classical potyviruses, including many conserved features. In phylogenetic analysis, SpMV could not be assigned to any of the known genera, but it showed the closest relationship to rymoviruses and common reed chlorotic stripe virus (CRCSV, unassigned). Sequence comparisons confirmed that a new genus should be established containing SpMV, CRCSV, and three Bermuda grass mosaic virus isolates, which are considered divergent strains of SpMV.

Complete genome sequence and construction of an infectious full-length cDNA clone of celery latent virus - an unusual member of a putative new genus within the Potyviridae.

Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11 519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40 % with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18 %) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.

Identification of a novel nanovirus in parsley.

Using next-generation sequencing to characterize agents associated with a severe stunting disease of parsley from Germany, we identified a hitherto undescribed virus. We sequenced total RNA and rolling-circle-amplified DNA from diseased plants. The genome sequence of the virus shows that it is a member of the genus Nanovirus, but it lacks DNA-U4. In addition to the seven genomic DNAs of the virus, we identified a second DNA-R and seven distinct alphasatellites associated with the disease. We propose the name "parsley severe stunt associated virus" (PSSaV) for this novel nanovirus.

Fluorescent labelling of Beet necrotic yellow vein virus and Beet soil-borne mosaic virus for co- and superinfection experiments in Nicotiana benthamiana.

Infectious full-length clones of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV), both genus Benyvirus, were used for fluorescent labelling with the objective to study their interaction in coinfection and superinfection experiments. Fluorescent labelling was achieved by replacing a part of the RNA2 encoded coat protein read-through domain with either GFP or mRFP fluorescent marker proteins. This resulted in a translational fusion comprising the coat and the fluorescent protein. The labelled viruses were infectious and moved systemically in Nicotiana benthamiana, producing wild-type-like symptoms. Virus particles could be observed by electron microscopy, demonstrating that the viral read-through domain is dispensable for particle formation. Coinfection experiments revealed a spatial separation of differentially labelled populations of both identical and different Benyvirus species after N. benthamiana agro-inoculation. Identical observations were obtained when Tobacco rattle virus (TRV) was differentially labelled and used for coinfection. In contrast, coinfections of BSBMV with Potato virus X (PVX) or TRV resulted in many co-infected cells lacking spatial separation. Micro-projectile co-bombardment of N. benthamiana leaves revealed that two differently labelled populations of the same virus co-infected only a few cells before starting to separate. In superinfection experiments with N. benthamiana, BSBMV and BNYVV were unable to establish a secondary infection in plants that were previously infected with BNYVV or BSBMV. Taken together, this is the first work to describe the interaction between two economically important Benyviruses using fluorescence-labelled full-length clones.

ICTV Virus Taxonomy Profile: Partitiviridae.

The Partitiviridae is a family of small, isometric, non-enveloped viruses with bisegmented double-stranded (ds) RNA genomes of 3-4.8 kbp. The two genome segments are individually encapsidated. The family has five genera, with characteristic hosts for members of each genus: either plants or fungi for genera Alphapartitivirus and Betapartitivirus, fungi for genus Gammapartitivirus, plants for genus Deltapartitivirus and protozoa for genus Cryspovirus. Partitiviruses are transmitted intracellularly via seeds (plants), oocysts (protozoa) or hyphal anastomosis, cell division and sporogenesis (fungi); there are no known natural vectors. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Partitiviridae, which is available at www.ictv.global/report/partitiviridae.

Complete genome sequence and construction of an infectious full-length cDNA clone of a German isolate of celery mosaic virus.

The complete genome sequence of a German isolate of celery mosaic virus (CeMV, a potyvirus) from Quedlinburg (DSMZ PV-1003) was determined (MF962880). This represents the second fully sequenced genome of this virus, along with a Californian isolate (HQ676607.1). The positive-sense single-stranded RNA is 10,000 nucleotides in length and shows the typical organization of potyviruses but has a shorter PIPO than CeMV California. In comparison to CeMV isolates from different origins, CeMV-Quedlinburg and isolates from the Netherlands (AF203531.1) and Aschersleben, Germany (AJ271087.1) show a NAG instead of DAG in the region of the coat protein responsible for aphid transmission. In this study the first infectious full-length clone of celery mosaic virus was obtained and the infectivity confirmed by Rhizobium radiobacter infiltration of Apium species.

Development of a molecular assay for the general detection of tospoviruses and the distinction between tospoviral species.

A Luminex xTAG-based assay for plant-infecting tospoviruses was developed. The test enables the detection of tospoviruses in general and the differentiation of the four important member species of this genus: Tomato spotted wilt virus, Impatiens necrotic spot virus, the proposed 'Capsicum chlorosis virus' and Watermelon silver mottle virus. The generic tospovirus primers used in this method are also applicable for detection of tospoviruses by basic RT-PCR. We also describe an economic alternative method for the distinction of the four tospoviruses mentioned and of additional member viruses, based on a restriction fragment length polymorphism (RFLP). The sophisticated Luminex xTAG technology allows the simultaneous detection of various targets. This study is part of a project that aims to develop a method for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material.

High Temperature Enhances the Resistance of Cultivated African Rice, Oryza glaberrima, to Bacterial Blight.

Rice bacterial blight (BB) is caused by Xanthomonas oryzae pv. oryzae and is responsible for substantial yield loss worldwide. Host resistance remains the most feasible control measure. However, pathogen variability leads to the failure of certain resistance genes to control the disease, and climate change with high amplitudes of heat predisposes the host plant to pathogen invasion. Due to pressure in natural selection, landrace species often carry a wide range of unique traits conferring tolerance of stress. Therefore, exploring their genetic background for host resistance could enable the identification of broad-spectrum resistance to combined abiotic and biotic stresses. Nineteen Oryza glaberrima accessions and O. sativa rice variety SUPA were evaluated for BB resistance under high temperature (35 and 31°C day and night, respectively) using 14 X. oryzae pv. oryzae strains originated from the Philippines. Under normal temperature, most of the accessions showed resistance to 9 strains (64.3%) and accession TOG6007 showed broad-spectrum resistance to 12 strains (85.7%). Under high temperature, most accessions showed a reduction in BB disease, whereas, accession TOG5620 showed disease reduction from all the X. oryzae pv. oryzae strains under high temperature. Molecular characterization using gene-based and linked markers for BB resistance genes Xa4, xa5, Xa7, xa13, and Xa21 revealed the susceptible alleles of Xa4, xa5, xa13, and Xa21 in O. glaberrima. However, no allele of Xa7 was detected among O. glaberrima accessions. Our results suggest that O. glaberrima accessions contain a BB resistance different from the Xa gene type. Genome-wide association mapping could be used to identify quantitative trait loci that are associated with BB resistance or combined BB resistance and high-temperature tolerance.

First full-length genome sequence of the polerovirus luffa aphid-borne yellows virus (LABYV) reveals the presence of at least two consensus sequences in an isolate from Thailand.

Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.

Pectobacterium aroidearum sp. nov., a soft rot pathogen with preference for monocotyledonous plants.

Several pectolytic bacterial strains, mainly isolated from monocotyledonous plants and previously identified as Pectobacterium carotovorum, were thought to belong to a novel species after several taxonomic analyses including DNA-DNA hybridization. In 16S rRNA gene sequence analyses, these strains had a similarity of >97.9 % to the 16S rRNA gene sequence of strains representing six other pectobacterial species and subspecies. These strains, represented by strain SCRI 109(T), also showed some unique chemotaxonomic features and quantitative differences in polar lipids, lipoquinones and fatty acids. A specific feature of strain SCRI 109(T) was the presence of DMK-8 lipoquinone, while the dominant fatty acids were the summed feature 3 (iso-C15 : 0 2-OH/C16 : 1ω7c), the unsaturated fatty acid C18 : 1ω7c and straight chain fatty acids, mainly C16 : 0. The DNA G+C content of strain SCRI 109(T) was 50.2 mol%. The taxonomic status of strain SCRI 109(T) and related strains in 16S rRNA gene sequence, chemotaxonomic, and physiological analyses was corroborated by the distinct clustering of these strains in multi-locus sequence analyses. It is proposed that these strains represent a novel species for which the name Pectobacterium aroidearum sp. nov. is proposed; the type strain is SCRI 109(T) ( = NCPPB 929(T) = LMG 2417(T) = ICMP 1522(T)).

Molecular characterization of five betacryptoviruses infecting four clover species and dill.

The family Partitiviridae includes plant (Alphacryptovirus and Betacryptovirus), fungal (Partitivirus) and protozoan (Cryspovirus) viruses with bisegmented dsRNA genomes and isometric virions. Cryptic viruses commonly occur in different plant species without causing any symptoms. So far, numerous sequences have been determined for viruses of the genus Alphacryptovirus, but no sequence is available for any assigned member of the genus Betacryptovirus. Following extraction, cloning and sequence analysis of double-stranded RNA in this study, we report the molecular properties of three assigned members of the genus Betacryptovirus, white clover cryptic virus 2, red clover cryptic virus 2 and hop trefoil cryptic virus 2, and two new putative betacryptoviruses found in crimson clover and dill. Betacryptoviruses share sequence motifs with members of the genus Partitivirus. In phylogenetic analyses, members of the genus Betacryptovirus formed a new sub-cluster within the clusters containing members of the genus Partitivirus. Our results provide evidence for a distinct evolutionary lineage of dsRNA viruses of plants and fungi.

The arms race between beet necrotic yellow vein virus and host resistance in sugar beet.

Beet necrotic yellow vein virus (BNYVV) causes rhizomania disease in sugar beet (Beta vulgaris), which is controlled since more than two decades by cultivars harboring the Rz1 resistance gene. The development of resistance-breaking strains has been favored by a high selection pressure on the soil-borne virus population. Resistance-breaking is associated with mutations at amino acid positions 67-70 (tetrad) in the RNA3 encoded pathogenicity factor P25 and the presence of an additional RNA component (RNA5). However, natural BNYVV populations are highly diverse making investigations on the resistance-breaking mechanism rather difficult. Therefore, we applied a reverse genetic system for BNYVV (A type) to study Rz1 resistance-breaking by direct agroinoculation of sugar beet seedlings. The bioassay allowed a clear discrimination between susceptible and Rz1 resistant plants already four weeks after infection, and resistance-breaking was independent of the sugar beet Rz1 genotype. A comprehensive screen of natural tetrads for resistance-breaking revealed several new mutations allowing BNYVV to overcome Rz1. The supplementation of an additional RNA5 encoding the pathogenicity factor P26 allowed virus accumulation in the Rz1 genotype independent of the P25 tetrad. This suggests the presence of two distinct resistance-breaking mechanisms allowing BNYVV to overcome Rz1. Finally, we showed that the resistance-breaking effect of the tetrad and the RNA5 is specific to Rz1 and has no effect on the stability of the second resistance gene Rz2. Consequently, double resistant cultivars (Rz1+Rz2) should provide effective control of Rz1 resistance-breaking strains. Our study highlights the flexibility of the viral genome allowing BNYVV to overcome host resistance, which underlines the need for a continuous search for alternative resistance genes.

Beet mosaic virus expression of a betalain transcription factor allows visual virus tracking in Beta vulgaris.

In the field of plant virology, the usage of reverse genetic systems has been reported for multiple purposes. One is understanding virus-host interaction by labelling viral cDNA clones with fluorescent protein genes to allow visual virus tracking throughout a plant, albeit this visualization depends on technical devices. Here we report the first construction of an infectious cDNA full-length clone of beet mosaic virus (BtMV) that can be efficiently used for Agrobacterium-mediated leaf inoculation with high infection rate in Beta vulgaris, being indistinguishable from the natural virus isolate regarding symptom development and vector transmission. Furthermore, the BtMV clone was tagged with the genes for the monomeric red fluorescent protein or the Beta vulgaris BvMYB1 transcription factor, which activates the betalain biosynthesis pathway. The heterologous expression of BvMYB1 results in activation of betalain biosynthesis genes in planta, allowing visualization of the systemic BtMV spread with the naked eye as red pigmentation emerging throughout beet leaves. In the case of BtMV, the BvMYB1 marker system is stable over multiple mechanical host passages, allows qualitative as well as quantitative virus detection and offers an excellent opportunity to label viruses in plants of the order Caryophyllales, allowing an in-depth investigation of virus-host interactions on the whole plant level.

Host range and molecular and ultrastructural analyses of Asparagus virus 1 pathotypes isolated from garden asparagus Asparagus officinalis L.

Asparagus samples were examined from growing areas of Germany and selected European as well as North, Central and South American countries. Overall, 474 samples were analyzed for Asparagus virus 1 (AV1) using DAS-ELISA. In our survey, 19 AV1 isolates were further characterized. Experimental transmission to 11 species belonging to Aizoaceae, Amarantaceae, Asparagaceae, and Solanaceae succeeded. The ultrastructure of AV1 infection in asparagus has been revealed and has been compared with the one in indicator plants. The cylindrical inclusion (CI) protein, a core factor in viral replication, localized within the cytoplasm and in systemic infections adjacent to the plasmodesmata. The majority of isolates referred to pathotype I (PI). These triggered a hypersensitive resistance in inoculated leaves of Chenopodium spp. and were incapable of infecting Nicotiana spp. Only pathotype II (PII) and pathotype III (PIII) infected Nicotiana benthamiana systemically but differed in their virulence when transmitted to Chenopodium spp. The newly identified PIII generated amorphous inclusion bodies and degraded chloroplasts during systemic infection but not in local lesions of infected Chenopodium spp. PIII probably evolved via recombination in asparagus carrying a mixed infection by PI and PII. Phylogeny of the coat protein region recognized two clusters, which did not overlap with the CI-associated grouping of pathotypes. These results provide evidence for ongoing modular evolution of AV1.

Detection of plum pox potyviral protein-protein interactions in planta using an optimized mRFP-based bimolecular fluorescence complementation system.

In previous studies, protein interaction maps of different potyviruses have been generated using yeast two-hybrid (YTH) systems, and these maps have demonstrated a high diversity of interactions of potyviral proteins. Using an optimized bimolecular fluorescence complementation (BiFC) system, a complete interaction matrix for proteins of a potyvirus was developed for the first time under in planta conditions with ten proteins from plum pox virus (PPV). In total, 52 of 100 possible interactions were detected, including the self-interactions of CI, 6K2, VPg, NIa-Pro, NIb and CP, which is more interactions than have ever been detected for any other potyvirus in a YTH approach. Moreover, the BiFC system was shown to be able to localize the protein interactions, which was typified for the protein self-interactions indicated above. Additionally, experiments were carried out with the P3N-PIPO protein, revealing an interaction with CI but not with CP and supporting the involvement of P3N-PIPO in the cell-to-cell movement of potyviruses. No self-interaction of the PPV helper component-proteinase (HC-Pro) was detected using BiFC in planta. Therefore, additional experiments with turnip mosaic virus (TuMV) HC-Pro, PPV_HC-Pro and their mutants were conducted. The self-interaction of TuMV_HCpro, as recently demonstrated, and the self-interaction of the TuMV_ and PPV_HC-Pro mutants were shown by BiFC in planta, indicating that HC-Pro self-interactions may be species-specific. BiFC is a very useful and reliable method for the detection and localization of protein interactions in planta, thus enabling investigations under more natural conditions than studies in yeast cells.