Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.     

Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector,phyllogen, induces phyllody

Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant‐pathogenic bacteria that transform plant floral organs into leaf‐like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma‐secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma‐infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two‐hybrid and in planta transient co‐expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin–proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody‐inducing gene family of ‘phyllogens’.     

Differential acquisition of chrysanthemum yellows phytoplasma by three leafhopper species

Chrysanthemum yellows (CY) phytoplasma has been transmitted with three leafhopper species: Euscelidius variegatus (Kirschbaum), Macrosteles quadripunctulatus (Kirschbaum) and Euscelis incisus (Kirschbaum): the first two species are reported as CY phytoplasma vectors for the first time. Leafhoppers were allowed to acquire the pathogen from the following source plants: Apium graveolens L., Catharanthus roseus L., Chrysanthemum carinatum Schousboe L. and C. frutescens L. DNA extracted from healthy or inoculative leafhoppers-exposed plants were analyzed by dot-blot and Southern hybridizations with a molecular probe constructed onto a fragment of European aster yellows phytoplasma DNA. The three leafhopper species were able to transmit CY phytoplasma after acquisition on chrysanthemum, but only M. quadripunctulatus and E. variegatus transmitted after feeding on periwinkle, and none acquired it from celery. All plant species tested were susceptible to CY, but while chrysanthemum and periwinkle were suitable for both inoculation and acquisition, celery did not seem to be a good source of phytoplasma for further inoculations. It is concluded that host plants influence leafhoppers' vectoring ability, possibly due to the different feeding behaviour of the insects on diverse plant species. Since CY, like several other phytoplasmas, can be transmitted by different insect species, it is likely that a close transmission specificity probably does not exist between phytoplasmas and their leafhopper vectors.     

Arranging the bouquet of disease: floral traits and the transmission of plant and animal pathogens

Several floral microbes are known to be pathogenic to plants or floral visitors such as pollinators. Despite the ecological and economic importance of pathogens deposited in flowers, we often lack a basic understanding of how floral traits influence disease transmission. Here, we provide the first systematic review regarding how floral traits attract vectors (for plant pathogens) or hosts (for animal pathogens), mediate disease establishment and evolve under complex interactions with plant mutualists that can be vectors for microbial antagonists. Attraction of floral visitors is influenced by numerous phenological, morphological and chemical traits, and several plant pathogens manipulate floral traits to attract vectors. There is rapidly growing interest in how floral secondary compounds and antimicrobial enzymes influence disease establishment in plant hosts. Similarly, new research suggests that consumption of floral secondary compounds can reduce pathogen loads in animal pollinators. Given recent concerns about pollinator declines caused in part by pathogens, the role of floral traits in mediating pathogen transmission is a key area for further research. We conclude by discussing important implications of floral transmission of pathogens for agriculture, conservation and human health, suggesting promising avenues for future research in both basic and applied biology.     

Transmission of sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector

Sugarcane white leaf disease is caused by plant pathogenic phytoplasmas that are transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). To determine whether there are other insect vectors that transmit this disease pathogen, leafhopper species in sugarcane, Saccharum officinarum L., fields in northeastern Thailand were monitored by using light traps. Sixty-nine leafhopper species from family Cicadellidae were found. Using nested polymerase chain reaction (PCR) with specific primers, a 210-bp amplified DNA fragment corresponding to phytoplasma associated with sugarcane white leaf disease was detected from 12 species of leafhoppers [Balclutha rubrostriata (Melichar), Balclutha sp., Bhatia olivacea (Melichar), Exitianus indicus Distant, Macrosteles striifrons Anufriew, Matsumuratettix hiroglyphicus (Matsumura), Recilia distincta (Motschulsky), Recilia dorsalis (Motschulsky), Recilia sp., Thaia oryzivora Ghauri, Yamatotettix flavovittatus Matsumura, and Xestocephalus sp.]. The percentage of individual infection with phytoplasma varied from 5% in B. olivacea to 35% in Xestocephalus sp. The most abundant leafhopper species, i.e., E. indicus, Y. flavovittatus, and M. hiroglyphicus were used in transmission tests to determine their vector status for the sugarcane white leaf phytoplasma transmission. Infected insects were reared on healthy plants and specific PCR followed by sequencing of the amplicons was used to determine whether the phytoplasma was transmitted to the plants. The results showed that both Y. flavovittatus and M. hiroglyphicus, but not E. indicus, can transmit sugarcane white leaf phytoplasma to healthy sugarcane plants. The transmission efficiency of M. hiroglyphicus (55%) was higher than that of Y. flavovittatus (45%). We conclude that Y. flavovittatus is a newly discovered vector for sugarcane white leaf disease, in addition to M. hiroglyphicus. These two species peak at different times of the year and therefore complement each other in the transmission of the phytoplasma. Because there are no known alternative host plants for the sugarcane white leaf, management of the disease will necessarily require the control of both Y. flavovittatus and M. hiroglyphicus.     

Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae) is the most important vector of sugarcane white leaf (SCWL) phytoplasma that significantly affects the sugarcane crop in Asia. Here, we aimed to study the characteristics of SCWL phytoplasma transmission by M. hiroglyphicus. To this end, the stylet penetration activities performed during the acquisition access period (AAP) and inoculation access period (IAP) were investigated by the direct current electrical penetration graph technique and confirmed by quantitative polymerase chain reaction (qPCR). Additionally, the latent period (LP) of SCWL phytoplasma in the vector was determined by qPCR and localised by fluorescent in situ hybridisation. The results indicated that the acquisition of SCWL phytoplasma occurred during phloem ingestion (waveform D), whereas its inoculation was associated with salivation into the phloem sieve element (waveform C). The minimum AAP was 15 min and the minimum duration of phloem ingestion was 2.35 min. The minimum LP of SCWL phytoplasma in the vector was at least 14 days; then, SCWL phytoplasma moved to the salivary glands of the insect, enabling the transmission of the pathogen to the host plants. The minimum IAP for a successful transmission of SCWL phytoplasma to the host plants was 11–12 min, with a minimum duration of salivation into phloem of 1.35 min. The female vectors had higher SCWL phytoplasma copy numbers than the male vectors, and displayed faster AAP, IAP, and LP. Overall, our findings provide important information related to the feeding behaviour of M. hiroglyphicus and its effect on the transmission of SCWL phytoplasma.     

Unique morphological changes in plant pathogenic phytoplasma-infected petunia flowers are related to transcriptional regulation of floral homeotic genes in an organ-specific manner

Abnormal flowers are often induced by infection of certain plant pathogens, e.g. phytoplasma, but the molecular mechanisms underlying these malformations have remained poorly understood. Here, we show that infection with OY-W phytoplasma (Candidatus Phytoplasma asteris, onion yellows phytoplasma strain, line OY-W) affects the expression of the floral homeotic genes of petunia plants in an organ-specific manner. Upon infection with OY-W phytoplasma, floral morphological changes, including conversion to leaf-like structures, were observed in sepals, petals and pistils, but not in stamens. As the expression levels of homeotic genes differ greatly between floral organs, we examined the expression levels of homeotic genes in each floral organ infected by OY-W phytoplasma, compared with healthy plants. The expression levels of several homeotic genes required for organ development, such as PFG, PhGLO1 and FBP7, were significantly downregulated by the phytoplasma infection in floral organs, except the stamens, suggesting that the unique morphological changes caused by the phytoplasma infection might result from the significant decrease in expression of some crucial homeotic genes. Moreover, the expression levels of TER, ALF and DOT genes, which are known to participate in floral meristem identity, were significantly downregulated in the phytoplasma-infected petunia meristems, implying that phytoplasma would affect an upstream signaling pathway of floral meristem identity. Our results suggest that phytoplasma infection may have complex effects on floral development, resulting in the unique phenotypes that were clearly distinct from the mutant flower phenotypes produced by the knock-out or the overexpression of certain homeotic genes.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.     

Proteinaceous effector discovery and characterization in filamentous plant pathogens

The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets.     

Detection,characterization and evolutionary aspects of S54LP of SP (SAP54 Like Protein of Sesame Phyllody): a phytoplasma effector molecule associated with phyllody development in sesame ( Sesamum indicum L.)

SAP54, an effector protein secreted by phytoplasmas has been reported to induce phyllody. S54LP of SP (SAP54 Like Protein of Sesame Phyllody), a SAP54 ortholog from phyllody and witches’ broom affected sesame (Sesamum indicum L.) was amplified, cloned and sequenced. Comparative sequence and phylogenetic analysis of diverse phytoplasma strains was carried out to delineate the evolution of S54LP of SP. The degree of polymorphism across SAP54 orthologs and the evolutionary forces acting on this effector protein were ascertained. Site-specific selection across SAP54 orthologs was estimated using Fixed Effects Likelihood (FEL) approach. Nonsynonymous substitutions were detected in the SAP54 orthologs’ sequences from phytoplasmas belonging to same (sub) group. Phylogenetic analysis based on S54LP of SP grouped phytoplasmas belonging to same 16SrDNA (sub) groups into different clusters. Analysis of selection forces acting on SAP54 orthologs from nine different phytoplasma (sub)groups, affecting plant species belonging to twelve different families across ten countries showed the orthologs to be under purifying (negative) selection. One amino acid residue was found to be under pervasive diversifying (positive) selection and a total of three amino acid sites were found to be under pervasive purifying (negative) selection. The location of these amino acids in the signal peptide and mature protein was studied with an aim to understand their role in protein–protein interaction. Asparagine residues (at positions 68 and 84) were found to be under pervasive purifying selection suggesting their functional importance in the effector protein. Our study suggests lack of coevolution between SAP54 and 16SrDNA. Signal peptide appears to evolve at a rate slightly higher than the mature protein. Overall, SAP54 and its orthologs are evolving under purifying selection confirming their functional importance in phytoplasma virulence.     

Molecular biology and pathogenicity of phytoplasmas

Phytoplasmas are a large group of plant‐pathogenic wall‐less, non‐helical, bacteria associated with diseases, collectively referred to as yellows diseases, in more than a thousand plant species worldwide. Many of these diseases are of great economic importance. Phytoplasmas are difficult to study, in particular because all attempts at culturing these plant pathogens under axenic conditions have failed. With the introduction of molecular methods into phytoplasmology about two decades ago, the genetic diversity of phytoplasmas could be elucidated and a system for their taxonomic classification based on phylogenetic traits established. In addition, a wealth of information was generated on phytoplasma ecology and genomics, phytoplasma–plant host interactions and phytoplasma–insect vector relationships. Taxonomically, phytoplasmas are placed in the class Mollicutes, closely related to acholeplasmas, and are currently classified within the provisional genus ‘Candidatus Phytoplasma’ based primarily on 16S rDNA sequence analysis. Phytoplasmas are characterised by a small genome. The sizes vary considerably, ranging from 530 to 1350 kilobases (kb), with overlapping values between the various taxonomic groups and subgroups, resembling in this respect the culturable mollicutes. The smallest chromosome, about 530 kb, is known to occur in the Bermuda grass white leaf agent ‘Ca. Phytoplasma cynodontis’. This value represents the smallest mollicute chromosome reported to date. In diseased plants, phytoplasmas reside almost exclusively in the phloem sieve tube elements and are transmitted from plant to plant by phloem‐feeding homopteran insects, mainly leafhoppers and planthoppers, and less frequently psyllids. Most of the phytoplasma host plants are angiosperms in which a wide range of specific and non‐specific symptoms are induced. Phytoplasmas have a unique and complex life cycle that involves colonisation of different environments, the plant phloem and various organs of the insect vectors. Furthermore, many phytoplasmas have an extremely wide plant host range. The dynamic architecture of phytoplasma genomes, due to the occurrence of repetitive elements of various types, may account for variation in their genome size and adaptation of phytoplasmas to the diverse environments of their plant and insect hosts. The availability of five complete phytoplasma genome sequences has made it possible to identify a considerable number of genes that are likely to play major roles in phytoplasma–host interactions. Among these, there are genes encoding surface membrane proteins and effector proteins. Also, it has been shown that phytoplasmas dramatically alter their gene expression upon switching between plant and insect hosts.     

Weedy hosts and prevalence of potential leafhopper vectors (Hemiptera: Cicadellidae) of a phytoplasma (16SrIX group) associated with Huanglongbing symptoms in citrus groves

Huanglongbing (HLB) is a severe citrus (Citrus spp.) disease associated with the bacteria genus Candidatus Liberibacter, detected in Brazil in 2004. Another bacterium was found in association with HLB symptoms and characterized as a phytoplasma belonging to the 16SrIX group. The objectives of this study were to identify potential leafhopper vectors of the HLB-associated phytoplasma and their host plants. Leafhoppers were sampled every other week for 12 mo with sticky yellow cards placed at two heights (0.3 and 1.5 m) in the citrus tree canopy and by using a sweep net in the ground vegetation of two sweet orange, Citrus sinensis (L.) Osbeck, groves infected by the HLB-phytoplasma in S?o Paulo state. Faunistic analyses indicated one Agalliinae (Agallia albidula Uhler) and three Deltocephalinae [Balclutha hebe (Kirkaldy), Planicephalus flavicosta (St?l), and Scaphytopius (Convelinus) marginelineatus (St?l)] species, as the most abundant and frequent leafhoppers (Hemiptera: Cicadellidae). Visual observations indicated an association of leafhopper species with some weeds and the influence of weed species composition on leafhopper abundance in low-lying vegetation. S. marginelineatus and P. flavicosta were more frequent on Sida rhombifolia L. and Althernantera tenella Colla, respectively, whereas A. albidula was observed more often on Conyza bonariensis (L.) Cronq. and B. hebe only occurred on grasses. DNA samples of field-collected S. marginelineatus were positive by polymerase chain reaction and sequencing tests for the presence of the HLB-phytoplasma group, indicating it as a potential vector. The association of leafhoppers with their hosts may be used in deciding which management strategies to adopt against weeds and diseases in citrus orchards.     

Development of a Mild Viral Expression System for Gain-Of-Function Study of Phytoplasma Effector In Planta

PHYL1 and SAP54 are orthologs of pathogenic effectors of Aster yellow witches’-broom (AYWB) phytoplasma and Peanut witches’-broom (PnWB) phytoplasma, respectively. These effectors cause virescence and phyllody symptoms (hereafter leafy flower) in phytoplasma-infected plants. T₀ lines of transgenic Arabidopsis expressing the PHYL1 or SAP54 genes (PHYL1 or SAP54 plants) show a leafy flower phenotype and result in seedless, suggesting that PHYL1 and SAP54 interfere with reproduction stage that restrict gain-of-function studies in the next generation of transgenic plants. Turnip mosaic virus (TuMV) mild strain (TuGK) has an Arg182Lys mutation in the helper-component proteinase (HC-Pro^R182K) that blocks suppression of the miRNA pathway and prevents symptom development in TuGK-infected plants. We exploited TuGK as a viral vector for gain-of-function studies of PHYL1 and SAP54 in Arabidopsis plants. TuGK-PHYL1- and TuGK-SAP54-infected Arabidopsis plants produced identical leafy flower phenotypes and similar gene expression profiles as PHYL1 and SAP54 plants. In addition, the leafy flower formation rate was enhanced in TuGK-PHYL1- or TuGK-SAP54-infected Arabidopsis plants that compared with the T₀ lines of PHYL1 plants. These results provide more evidence and novel directions for further studying the mechanism of PHYL1/SAP54-mediated leafy flower development. In addition, the TuGK vector is a good alternative in transgenic plant approaches for rapid gene expression in gain-of-function studies.     

Beet leafhopper (Hemiptera: Cicadellidae) transmits the Columbia Basin potato purple top phytoplasma to potatoes, beets, and weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including 'Russet Burbank', 'Ranger Russet', 'Shepody', 'Umatilla Russet', 'Atlantic', 'FL-1879', 'FL-1867', and 'FL-1833', were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd's purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.     

Vector status of three leafhopper species for Australian lucerne yellows phytoplasma

Abstract   The leafhoppers Orosius argentatus (Evans), Austroagallia torrida (Evans) and Batracomorphus angustatus (Osborn) were used in transmission tests to determine their vector status for the phytoplasma associated with Australian lucerne yellows (ALuY). Caged, seed-grown lucerne plants were monitored for foliar symptom expression after feeding by leafhoppers transferred from ALuY symptomatic lucerne plants. Twelve of 25 plants developed phytoplasma disease-like symptoms including stunting and yellowing. The most pronounced foliar symptoms were displayed by five plants that had been fed on by O. argentatus and four plants that had been fed on by A. torrida. One plant, fed on by O. argentatus , showed the distinctive root symptoms of ALuY . A phytoplasma was identified by electron microscopy in two plants fed on by O. argentatus and one by A. torrida. For each group of plants that had been fed on by a single leafhopper species, one plant was phytoplasma positive as determined by the polymerase chain reaction (PCR) using universal primers. The phytoplasma detected by PCR in the plant fed on by A. torrida was identified by restriction fragment length polymorphism (RFLP) analysis as the tomato big bud (TBB) phytoplasma. The PCR product from two plants fed on by B. angustatus and O. argentatus were too faint for RFLP analysis. PCR assays were conducted on DNA extracted from the head and thorax of each leafhopper species from transmission tests and from field-collected insects, but no phytoplasma DNA was detected. These findings suggest O. argentatus is a vector of the ALuY pathogen and A. torrida is a vector of the TBB phytoplasma.     

A gravity model for the spread of a pollinator-borne plant pathogen

Many pathogens of plants are transmitted by arthropod vectors whose movement between individual hosts is influenced by foraging behavior. Insect foraging has been shown to depend on both the quality of hosts and the distances between hosts. Given the spatial distribution of host plants and individual variation in quality, vector foraging patterns may therefore produce predictable variation in exposure to pathogens. We develop a "gravity" model to describe the spatial spread of a vector-borne plant pathogen from underlying models of insect foraging in response to host quality using the pollinator-borne smut fungus Microbotryum violaceum as a case study. We fit the model to spatially explicit time series of M. violaceum transmission in replicate experimental plots of the white campion Silene latifolia. The gravity model provides a better fit than a mean field model or a model with only distance-dependent transmission. The results highlight the importance of active vector foraging in generating spatial patterns of disease incidence and for pathogen-mediated selection for floral traits.     

Rapid Detection by Genomic Amplification of Loofah Witches' Broom Phytoplasma in Leafhopper Vectors

Loofah (Luffa cylindrical) is a common vegetable crop in Taiwan and other Asian countries. Reported here is a novel rapid approach for detecting loofah witches’ broom (LfWB) phytoplasma in single leafhoppers. Field samples of suspected diseased plants and potential vectors from southern Taiwan were processed to test for the presence of the LfWB phytoplasmas using both strain‐specific DNA hybridization (DH) and polymerase chain reaction (PCR) assays. The commonest pathogen causing loofah disease in southern Taiwan is LfWB phytoplasma. Leafhoopers collected at nine locations near LfWB‐infected plants were found to be positive for LfWB by PCR / DH at an incidence of 28.5–40.0%. Of the different leafhopper species tested, only Hishimonus concavus was positive for LfWB, suggesting that H. concavus is a natural vector of LfWB in Taiwan. Using our proposed primers in this PCR assay, a single LfWB‐infected leafhopper can be detected rapidly and directly.