Lotus japonicus ARPC1 Is Required for Rhizobial Infection1[W] Md Shakhawat Hossain 2, Jinqiu Liao, Euan K. James, Shusei Sato, Satoshi Tabata, Anna Jurkiewicz, Lene H. Madsen, Jens Stougaard, Loretta Ross, and Krzysztof Szczyglowski* Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario, Canada N5V 4T3 (M.S.H., J.L., L.R., K.S.); The James Hutton Institute, Invergowrie, Dundee DD2 5DA, United Kingdom (E.K.J.); Kazusa DNA Research Institute, Kisarazu, Chiba 292–0818, Japan (S.S., S.T.); Department of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling, Aarhus University, 8000 Aarhus C, Denmark (A.J., L.H.M., J.S.); and Department of Biology, University of Western Ontario, London, Ontario, Canada N6A 5BF (K.S.)
Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nckassociated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.
Unlike the majority of angiosperms, leguminous plants have a capacity to host nitrogen-fixing rhizobia within living cells of root-derived organs, called nodules (Doyle, 2011). In most cases rhizobia enter plant roots through root hairs in a process that is carefully monitored by the host (Sprent and James, 2007; Murray, 2011). Local degradation of the root hair cell wall, which in Lotus japonicus requires activation of the plant pectate lyase (Xie et al., 2012), is thought to provide access for bacteria to the plasma membrane surface, from where an infection thread (IT) is initiated. The IT originates as a plasma membrane invagination from a discrete position within the curled root hair tip where a dominant microcolony of rhizobia has been captured (Jones et al., 2007). This provides a 1 This work was supported by grants from Agriculture and AgriFood Canada Crop Genomics Initiative and National Science and Engineering Research Council of Canada (Natural Sciences and Engineering Research Council of Canada grant no. 3277A01 to K.S.). A.J., L.H.M., and J.S. were supported by the Danish National Research Foundation. 2 Present address: Division of Plant Sciences, 201 Life Science Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211. * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Krzysztof Szczyglowski (
[email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.112.202572
plant-made conduit for rhizobia to penetrate the root interior. The IT extends through a tip growth-like mechanism, leading to its transcellular progression through the root hair and subsequently into the root cortical cells (Fournier et al., 2008), where subtending cell divisions for nodule primordia formation have been initiated. Once within the division center, bacteria are released from branched ITs into the cytoplasm of nodule primordia cells and become organelle-like structures encapsulated inside double-membrane-bound symbiosome compartments. There, they undergo final differentiation and initiate nitrogen fixation, thus establishing functional root nodule symbiosis (Kereszt et al., 2011). Perception of bacterially derived lipochitinoligosaccharide signaling molecules, known as nodulation or Nod factors (NFs; Lerouge et al., 1990) is required to initiate the infection process. In L. japonicus this necessitates two Lys-motif receptor-like kinases, namely NOD FACTOR RECEPTOR1 and NOD FACTOR RECEPTOR5 (Madsen et al., 2003; Radutoiu et al., 2003). Downstream from NF perception, elements of the so-called common symbiosis pathway (Kistner et al., 2005; Groth et al., 2010), which is also essential for intracellular accommodation of arbuscular mycorrhiza (AM) fungi inside the host root (Duc et al., 1989; Kistner et al., 2005), participate in the activation of calcium (Ca2+) signaling. This includes Ca2+ influx, which is associated with depolarization of plasma membrane, and rapid oscillation of Ca2+ concentrations in nucleoplasm, referred to as Ca2+ spiking (Oldroyd and
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Downie, 2006; Sieberer et al., 2009, 2012). The Ca2+ and calmodulin-dependent receptor kinase is thought to translate these early cellular responses into downstream reprogramming of the plant cells for infection and nodule organogenesis (Kistner et al., 2005; Høgslund et al., 2009; Singh and Parniske, 2012). Alterations in the host plant cytoskeleton associates with these early NF-dependent signaling events. The observation that leguminous plants carrying deleterious mutations in NCK-ASSOCIATED PROTEIN1 (NAP1) and/or 121F-SPECIFIC P53 INDUCIBLE RNA (PIR1) are defective in IT initiation and growth (Yokota et al., 2009; Miyahara et al., 2010) extended earlier data linking the dynamic rearrangement of the host plant cytoskeleton with the infection process (Timmers, 2008 and refs. therein). In other organisms, NAP1 and PIR1 are components of a pentameric regulatory complex that includes suppressor of cAMP receptor/WASP-family verpolin homologous protein (SCAR/WAVE), the major class 1 actin nucleation-promoting factor in plants (Campellone and Welch, 2010). This complex has been shown to act by linking various external or endogenous cues with the ACTIN-RELATED PROTEIN2/3 (ARP2/ 3) complex, the major nucleator of Y-branched actin filament networks in plants (Goley and Welch, 2006). A key role for the ARP2/3 complex has been demonstrated for several processes, such as cell migration and adhesion, membrane remodeling during cell trafficking events, disease development, as well as overall viability in some organisms (Goley and Welch, 2006). Strong infection defects in legumes’ nap1 and pir1 mutants predict an additional role for ARP2/3 during root colonization by symbiotic rhizobia. Intriguingly, several enteroinvasive bacterial pathogens, such as Salmonella enterica and Listeria monocytogenes, are known to exploit the host cell ARP2/3-dependent membrane ruffling for their entry and/or cell to cell motility (Cossart and Sansonetti, 2004). Considering the previously hypothesized pathogenic origin of symbiotic bacteria (Deakin and Broughton, 2009), a question arises as to whether a similar mechanism operates during symbiotic entry. In this study we demonstrate that the L. japonicus ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) gene, which encodes a presumed component of the L. japonicus heptameric ARP2/3 nucleator, is indeed essential for intracellular accommodation of rhizobial bacteria but is dispensable for arbuscular mycorrhiza (AM) symbiosis.
RESULTS A Novel L. japonicus Locus Required for Symbiotic Infection
A screen for genetic suppressors of the L. japonicus har1-1 hypernodulation phenotype identified a mutant line, called LjB32-BB (Murray et al., 2006). Like nap1 and pir1, LjB32-BB formed mostly uncolonized, 918
white nodules (Fig. 1) and distorted trichomes (see below). Mapping experiments positioned the LjB32BB locus to a unique location on L. japonicus chromosome 6, which was subsequently confirmed by identification of the causative gene. For reasons described below this gene was named as ARPC1 and the corresponding mutant allele is referred to hereafter as arpc1 (see below). In a mapping population maintaining the har1-1 genetic background (see “Materials and Methods”), arpc1 segregated as a single recessive locus, with 851 out of 3,433 plants showing the mutant symbiotic phenotype, consistent with a 3:1 ratio (x2 = 0.0817). The arpc1 har1-1 double mutant was subsequently backcrossed to wild-type L. japonicus ecotype Gifu and the har1-1 locus was segregated away to establish a single arpc1 mutant that was used in subsequent phenotypic analyses.
arpc1 Aborts Bacterial Infection
Twenty-one days after inoculation (dai), large pink nodules that were fully colonized by Mesorhizobium loti and a few nodule primordia were present on roots of wild-type L. japonicus plants (Figs. 1, A, B, and H and 2A). In contrast, arpc1 formed many uncolonized white nodules and nodule primordia (Fig. 1, C and H). M. loti accumulated on the surface of arpc1 nodules (Figs. 1D and 2B) and only very rarely managed to penetrate further (see below); pink, wild-type-like nodules were observed only sporadically on the mutant roots (0.5 6 0.22/plant). The mutant features were correlated with defects in root hair responses to NF application and infection by M. loti. In a liquid medium supplemented with M. loti NF (see “Materials and Methods”), wild-type root hairs showed typical deformation responses (Supplemental Fig. S1, A and B), whereas root hairs of the arpc1 susceptible root zone were short and unresponsive (Supplemental Fig. S1C). Elongated root hairs that were present higher up on arpc1 roots were also unresponsive to NF treatment (Supplemental Fig. S1D). When grown in soil, numerous microcolonies and ITs were present on wild-type roots 7 dai (Fig. 1, E and I), whereas only a few of these events could be found on arpc1 roots (Fig. 1I). Unlike in liquid or on agar plates, root hair elongation in arpc1 was not significantly affected under soil growth conditions (see below) but M. loti was only sporadically observed to accumulate at or within tips of small root hairs (Fig. 1F). When ITs were initiated, they were prematurely terminated within root hairs and often appeared disintegrated (Fig. 1G). In rare cases, when bacteria managed to enter the arpc1 nodule interior, localized infection regions were formed (Fig. 2, C–E). The bacteria were confined to sporadic cortical ITs, which often appeared swollen (Figs. 2E and 3A). In wild-type nodules, normally Plant Physiol. Vol. 160, 2012
ARPC1 Regulates Symbiotic Entry
Figure 1. Phenotype of the arpc1 mutant. A, Wild-type L. japonicus nodules at 21 dai are fully colonized by M. loti (B). The arpc1 mutant of the same age develops small, uncolonized white nodules (C) where bacteria are restricted to the nodule surface (D). E, Root hair IT-dependent colonization of wild-type L. japonicus root by M. loti. F, Colonization of short root hairs by M. loti and an example of disintegrated IT (G) in arpc1. M. loti strain NZP2235 tagged with a constitutive hemA:lacZ reporter gene fusion was used for inoculation and roots were stained for b-galactosidase activity (dark blue) to reveal the location of bacteria (sections B, D, and E–G) and to quantify the nodulation and infection events. H, Scores of various nodulation events 21 dai. I, Microcolonies (MC) and root hair ITs; Combined numbers of intact and aborted ITs per root in the wild-type and arpc1 mutant are given for 7 dai. Ten individuals were scored for each genotype (H) or infection category (I) and averages 6SE are given. Asterisks (*) indicate statistically significant differences.
formed symbiosomes were present at this stage (Fig. 3B). Rare nodule cells that appeared fully infected were detected in arpc1 (Fig. 2E), however, closer inspection revealed that they were almost entirely filled with a tangled IT (Fig. 3C). This most likely gave rise to infected regions of the arpc1 nodule cortex (Fig. 2, C and D), where a few enlarged nodule cells contained released bacteria but were mostly deprived of typical cellular structures, including symbiosomes (Fig. 3D). We have interpreted this to be the result of ongoing cellular degradation of the IT-filled cells in the arpc1 mutant. In contrast to defective bacterial infection, the arpc1 mutant formed wild-type AM symbiosis (Fig. 3, E and F).
Identification of the Causative Mutation
The ARPC1 locus was positioned to a genetic interval of approximately 0.4 centimorgan on L. japonicus chromosome 6, between simple sequence repeat markers TM0836 and TM0331 (Supplemental Fig. S2, A and B). Based on overall phenotypic similarity between Plant Physiol. Vol. 160, 2012
arpc1 and nap1 and pir1, we reasoned that the ARPC1 locus is likely to encode a protein with a function related to the plant cytoskeleton. We have used the available sequence information to search for such a protein. One of the genes identified within the delimited region, namely ARPC1 (Supplemental Fig. S2C), was predicted to encode a protein (Supplemental Fig. S2D) with a significant similarity to the Arabidopsis (Arabidopsis thaliana) ARPC1A and 1B (see below). Sequencing of the ARPC1 locus from the mutant and wild-type Gifu revealed a single nucleotide polymorphism, reflected by the substitution of C1809 to T at the ARPC1 locus (Supplemental Fig. S2C). Using 55 mutants derived from the mapping population and the arpc1-specific marker (see “Materials and Methods”), cosegregation of the nodulation/infection and trichome mutant phenotypes with the arpc1 mutation was observed. Thus, the identified L. japonicus gene (ARPC1) was considered a viable candidate for the mutated locus in arpc1. This was further confirmed by performing in planta complementation experiments, as the ARPC1 gene complemented the arpc1 mutant root hair and nodulation phenotypes (Supplemental Fig. S3). Cumulatively, these results showed that the 919
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Figure 2. arpc1 is defective in infection by M. loti. A, A semithin (approximately 1 mm) section of a typical wild-type nodule showing infected cells (blue). B, A thick section (approximately 35 mm) through a typical arpc1 nodule showing lack of bacterial colonization; note that bacteria are restricted to the nodule surface (blue and arrow). C and D, A rare, partially colonized nodule in arpc1 shown as thick (C; approximately 35 mm) and semithin (D; 1 mm) sections. E, Magnification of a region delimited by the rectangle in section D, showing enlarged cortical ITs and three nodule infected cells (IC).
correct gene was identified and that the arpc1 mutation was also responsible for the mutant root hair phenotype. ARPC1 Is a WD40 Repeat-Containing Protein
The ARPC1 mRNA was determined to be 1,443 bp. It contained a predicted open reading frame of 376 amino acids, with an estimated ARPC1 protein product of 42.2 kD. A presumed ATG initiation codon was preceded by a 94-bp-long 59 untranslated region containing no other in frame ATG codons. In the genomic context, the ARPC1 mRNA sequence was distributed among 15 exons (Supplemental Table S1), which was identical to the organization of ARPC1-encoding genes from Arabidopsis and Physcomitrella patens (Harries et al., 2005). Like all ARPC1 proteins identified to date, the L. japonicus ARPC1 was predicted to have seven WD40 repeats (Supplemental Fig. S2D); these are thought to form a seven-bladed b-propeller structure (Robinson et al., 2001). The arpc1 mutation was located 920
within the second exon and led to a premature termination of the open reading frame, by replacing the CAA codon for Q51 to a TAA stop codon (Supplemental Fig. S2D). The ARPC1 protein showed the highest homology (94% identity and 97% similarity) to an as yet uncharacterized WD40-repeat-containing protein from Glycine max (Supplemental Fig. S4). Importantly, ARPC1 homologs from a wide range of organisms shared between 35% and 79% amino acid identity with L. japonicus ARPC1 (Supplemental Fig. S4). Based on these observations and the arpc1 mutant phenotype, we concluded that ARPC1 likely encodes the subunit 1 of the presumed L. japonicus ARP2/3 complex. ARPC1 Is Ubiquitously Expressed in L. japonicus
The ARPC1 transcript was present in all tissues tested, including nodules, with the highest steady-state level in developing seed pods (Fig. 4A). To gain insight into localization of the ARPC1 gene promoter activity, Plant Physiol. Vol. 160, 2012
ARPC1 Regulates Symbiotic Entry
Figure 3. Microscopy of infections in wild-type and arpc1. A to D, TEM images of ultrathin (80 nm) sections of nodules. A, A cortical IT traversing a cell boundary in arpc1. B, Symbiosomes (s) containing bacteroids (b) within an infected cell of wild-type L. japonicus nodules. C, A portion of a rare infected cell in arpc1 nodule filled with a tangled IT. D, A portion of an extensive infection region (see Fig. 2, C–E) in a partially colonized nodule of arpc1, showing M. loti present within a hugely enlarged cortical cell; note the absence of typical cellular structures, including symbiosomes, which likely indicates the ongoing cellular degradation process. Representative fragments of L. japonicus wild-type (E) and arpc1 (F) roots successfully colonized by the AM fungus, Glomus intraradices.
transgenic hairy roots carrying the ARPC1pro:GUS reporter gene construct were analyzed (Fig. 4, B–D). The GUS activity was detected throughout the entire root system, including root hairs and nodules (Fig. 4, B–D). Nonsymbiotic Phenotype of arpc1
Compared with elongated filamentous structures of wild-type plants, arpc1 had distorted trichomes that were deformed and shorter (Fig. 5, A–F). arpc1 also showed mutant seed pod (Fig. 5G) and root hair phenotypes (Fig. 6); though root hairs initiated as in the wild type (Fig. 6, A and B) their elongation was defective under rapid growth-stimulating conditions. Thus, when grown on the surface of vertically positioned agar plates, arpc1 roots had noticeably shorter and more variable-length root hairs (Fig. 6, C and D). This was not associated with any apparent impairment in the root elongation (Fig. 7A). Root hairs of soilgrown arpc1 plants were indistinguishable from those of wild-type plants (Fig. 6, E and F). The shoot development was unaffected in uninoculated arpc1 grown in the presence of combined nitrogen. Twenty-one days after sowing, the average shoot lengths of the wild type and arpc1 were 22.8 6 0.32 mm and 22.1 6 0.26 mm, respectively; however, arpc1 formed shorter pods (Figs. 5G and 7B). The average Plant Physiol. Vol. 160, 2012
length of hypocotyls in dark-grown plants (Fig. 7C) and in vitro pollen tube formation in arpc1 were not significantly different from wild-type plants (Fig. 7, D and E). Furthermore, no significant changes in the shape of arpc1 embryonic leaf pavement cells could be detected in comparison with wild-type cotyledons (Fig. 7, F and G). Actin Structure in arpc1
The actin structure in the wild type and the arpc-1 mutant was analyzed by imaging live root hairs in transgenic hairy roots expressing the 35S:GFP-ABD2GFP F-actin reporter (Wang et al., 2008). Changes to the F-actin structure were minor and observable only in short root hairs of arpc1 (,20–30 mm; n = 10). Actin filaments in these root hairs were organized in more transverse and less longitudinally aligned cables (Fig. 8B), which resembled the mutant F-actin organization of L. japonicus nap1 and pir1 root hairs (Yokota et al., 2009). In rare cases, formation of thick actin filaments was also observed in the mutant (Fig. 8C). However, longer arpc1 root hairs (.20–30 mm) showed wildtype-like arrangement of actin filaments, as reflected by the presence of actin cables that run predominantly parallel to the long axis of root hairs (Fig. 8, A and D), which also paralleled observations made for L. 921
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molecules, including viruses and bacteria (Pollitt and Insall, 2009). We show here that the arpc1 mutation, which affects a predicted ARPC1 subunit of a presumed L. japonicus ARP2/3 complex, almost completely prevents root infection by M. loti and inflicts several developmental defects on processes involving expansion cell growth. Together with the previously reported impaired symbiotic infection phenotypes of L. japonicus nap1 and pir1 (Yokota et al., 2009), our data indicate that ARPC1 and, by inference a SCAR/ WAVE-ARP2/3 pathway, plays an essential role during intracellular colonization of roots by symbiotic bacteria.
ARPC1 Is Required during Expansion Growth
Figure 4. The ARPC1 gene is expressed ubiquitously in L. japonicus. A, Quantitative RT-PCR analysis of the ARPC1 mRNA in various L. japonicus tissues; UNR, 14-d-old uninoculated roots; 4D, 7D, 10D, and 12D, roots harvested at the given dai with M. loti; 21D, nodules harvested 21 dai. B to D, Histochemical localization of GUS activity in transgenic hairy roots, as mediated by the ARPC1 promoter. The ARPC1pro:GUS expression construct was introduced by Agrobacterium rhizogenes-mediated transformation and the resulting transgenic hairy roots were analyzed 14 dai. B, A global view of a representative segment of the transgenic hairy root expressing the ARPC1pro:GUS reporter construct. Close-ups showing expression in the root hairs (C) and developing nodule (D); tissues were stained for 12 h at 37˚C.
japonicus nap1 and pir1 mutants. Due to a strong root hair defect in arpc1 under plate-growth conditions and the resulting lack of responsiveness to NF application (Yokota et al., 2009), the impact of the arpc1 mutation on the NF-dependent actin cytoskeleton rearrangements was not investigated. DISCUSSION The SCAR/WAVE-ARP2/3 Pathway Is Essential for Bacterial Entry
Dynamic remodeling of the actin cytoskeleton partakes in many cellular processes, including intracellular transport and endocytic uptake of a variety of 922
Like Arabidopsis mutants in predicted ARP2/3 subunit genes, including ARP2, ARP3, ARPC2, and ARPC5 (Le et al., 2003; Li et al., 2003; Mathur et al., 2003a, 2003b; El-Din El-Assal et al., 2004), arpc1 did not affect pollen tube formation but impaired trichome development and limited root hair elongation under conditions that stimulated rapid growth. Unlike the Arabidopsis mutants, however, elongation of darkgrown hypocotyls and the shape formation in pavement cells of embryonic leaves were not significantly affected by the arpc1 mutation. arpc1 also showed short and collapsed seed pods, phenotypes not reported for Arabidopsis ARP2/3 mutants but described for L. japonicus nap1 and pir1 (Yokota et al., 2009). Species or ARPC1 subunit-specific effects could be the reason for some of these phenotypic differences, but aside from arpc1 no other reports on higher plant ARPC1 mutants are currently available, and thus this will have to await further verification. It is important to note, however, that unlike in P. patens, where inactivation of ARPC1 by RNAi causes significant defects in tip growth and cell division patterning (Harries et al., 2005), Lotus and Arabidopsis tip growing pollen tubes and root hairs, appear relatively insensitive to mutations in ARP2/3 complex subunits under normal growth conditions. The root hair elongation defect observed under rapid growth conditions in aprc1 was more severe than the corresponding effect of the L. japonicus nap1 and pir1 mutations (Yokota et al., 2009). The presence of SCARE/WAVE-independent ARP2/3 activators (Galletta et al., 2008) might have contributed to lessening the impact of nap1 and pir1 on the root hair development. Nevertheless, a rather minor alteration to F-actin cytoskeleton in arpc1 paralleled the corresponding nap1 and pir1 phenotypes. The Y-branched filaments constitute a small fraction of the total actin cytoskeleton and this might explain why more conspicuous effects were not observed in these mutants. Unlike in yeast (Saccharomyces cerevisiae), Caenorhabditis elegans, Drosophila spp., or human HeLa cells, where mutations in ARP2/3 subunits lead to drastic developmental defects or lethality, the function of the ARP2/3 Plant Physiol. Vol. 160, 2012
ARPC1 Regulates Symbiotic Entry
Figure 5. arpc1 has distorted trichome and mutant seed pod phenotypes. A, B, and D, Trichomes of wild-type L. japonicus formed on abaxial surface of the leaf midvein (A and D) and fully expanded flowers (B). C, E, and F, Corresponding, short trichomes of arpc1. Note that A and F represent SEM images of the wild-type and arpc1 leave trichomes, respectively, which are shown at different magnifications (see scales). G, Seed pods of the wild type (left) and arpc1 (right).
complex appears nonessential in plants (Goley and Welch, 2006) and the arpc1 phenotype is consistent with this notion. This might reflect some level of redundancy, possible involving other actin nucleators. arpc1 Aborts Root-Hair-Dependent and Root-Hair-Independent Infections
The arpc1 mutation significantly impaired root hair IT formation and/or maintenance, showing that ARPC1 is essential for this process. On the other hand, intracellular colonization of L. japonicus roots by M. loti can proceed by different modes, including the epidermal root hair IT-independent crack-entry-like mechanisms (Karas et al., 2005; Groth et al., 2010; Madsen et al., 2010; Kosuta et al., 2011). Furthermore, analysis of the L. japonicus roothairless mutant showed that the colonization of nodules can occur in a total absence of root hairs (Karas et al., 2005, 2009). As the majority of arpc1 nodules remained uncolonized by M. loti, the presence of the intact ARPC1 protein could be pertinent to different modes of intracellular root colonization by M. loti. Plant Physiol. Vol. 160, 2012
The sporadic presence of epidermal ITs in arpc1 could be due to a partially redundant function of ARP2/3 complex members or other actin nucleators, such as formins (Chesarone et al., 2010). Perhaps, this could also explain the occurrence of cortical ITs in rare, partially colonized arpc1 nodules, although we cannot entirely rule out the possibility that requirements for ARPC1 are different in these two cell types. Nevertheless, the apparent lack of normal release of bacteria from cortical ITs inside nodule cells suggests that ARPC1 might also be essential at this key stage during symbiosis. Role of ARPC1 and Actin Cytoskeleton during Infection
Reprogramming of plant cells for bacterial endosymbiosis involves cytoskeleton reorganization. The role of ARPC1 in this process is indirectly inferred here by the fact that NAP1 and PIR1, which regulate the ARP2/3 complex, are essential for inciting rapid rearrangement of actin filaments in L. japonicus (Yokota et al., 2009). The impairment in trichome development, the root hair deformation response, microcolony 923
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by longitudinal arrays of dense MTs (Timmers, 2008). Based on the analysis of the Arabidopsis DISTORTED genes, polar growth directionality was suggested to be guided by the cortical actin organization-dependent MT arrangement (Schwab et al., 2003; Saedler et al., 2004). Although evidence is not provided by our study, it is possible that the ARP2/3-dependent Y-branched actin networks participate in the initial selection of the IT initiation site and/or subsequent guidance of the cytoplasmic MTs, such that polarity fixation is established and maintained.
At Least Two Pathways Contribute to the IT-Dependent Infection
Figure 6. arpc1 affects normal elongation of root hairs under rapid growth conditions. Plants were grown on the surface of agar (A–D) or in soil (E and F) and representative segments at the root hair emergence (A and B) and mature root hair (C–F) zones were photographed 7 d after sowing. A, C, and E, Wild-type L. japonicus. B, D, and F, arpc1.
capturing, and IT initiation and/or maintenance in nap1 and pir1 (Yokota et al., 2009), together with our observations of similar defects in Ljarpc1, further suggests that the regulation of actin filament networks, as mediated by the SCARE/WAVE-ARP2/3 pathway, is essential in these processes. The importance of the host plant microtubular (MT) cytoskeleton is suggested by its recruitment to the site of IT initiation and subsequent molding of the IT body 924
Neither ARPC1 nor NAP1 or PIR1 are required for nodule primordia organogenesis. The wild-type AM phenotypes of L. japonicus arpc1 and also nap1 and pir1 mutants (Yokota et al., 2009) show that these proteins are also not essential for intracellular accommodation of the symbiotic fungi. The latter highlights a clear distinction between the two symbiotic entry mechanisms, which otherwise share a requirement for the host plant common symbiosis pathway (Held et al., 2010). Together with NAP1 and PIR1 (Yokota et al., 2009; Miyahara et al., 2010), our data indicate that ARPC1 and, by inference a SCAR/WAVE-ARP2/3 pathway must have been coopted during evolution to specifically mediate bacterial entry. Interestingly, the WASPfamily nucleation-promoting factors are known to be manipulated by a variety of mechanisms during infection by intracellular pathogens, such as Salmonella enterica, which leads to activation of the ARP2/3 complex and actin-dependent internalization of the bacterium inside nonphagocytic mammalian cells (Patel and Galán, 2005). An important challenge for future investigations will be to define how rhizobial signaling activates the SCAR/WAVE-ARP2/3 pathway, and how this relates to other functions that promote symbiotic infection (Haney and Long, 2010; Lefebvre et al., 2010). The common symbiosis Ca2+ and calmodulin-dependent receptor kinase/CYCLOPS complex was proposed to be responsible for cross signaling with an infection pathway that includes NAP1 and PIR1 (Madsen et al., 2010). This could constitute a mechanism by which the SCAR/WAVE-ARP2/3 and common symbiosis pathways are integrated to mediate symbiotic entry of bacteria. CONCLUSION
We demonstrate here that the L. japonicus mutant line arpc1, which originated from a screen for genetic suppressors of the har1-1 hypernodulation (Murray et al., 2006), carries a deleterious mutation in the gene encoding a predicted L. japonicus ARPC1 ortholog, one of the seven polypeptides that in other organisms comprise the ARP2/3 nucleator and are conserved in Plant Physiol. Vol. 160, 2012
ARPC1 Regulates Symbiotic Entry
Figure 7. arpc1 affects seed pod growth but does not impair root, hypocotyl, pollen tubes, or pavement cell development. A and C, Plants were grown on the surface of agar for 5 and 7 d in the dark upon which the length of roots (A; n = 20) and hypocotyls (C; n = 50) were measured. The average seed pod length (B) is given for n = 25. Mean values 6SE are given in each graph. In vitro pollen germination in the wild type (D) and arpc1 (E). SEM images of pavements cells in cotyledons of L. japonicus wild-type (F) and arpc1 mutant (G) are shown. Note that although no dramatic differences in the cell shape were detected, pavement cells of the wild type appear to have somewhat more irregular shape.
nearly all eukaryotic organisms. The arpc1 mutation almost entirely prevented root colonization of L. japonicus by its natural microsymbiont, M. loti, but had no negative effect on AM-fungi colonization. Like nap1 and pir1, arpc1 was also defective in trichome and seed pod development and had a detrimental effect on root hair elongation under conditions of rapid
growth, indicating that the ARP2/3-dependent dynamic nucleation of the actin cytoskeleton is indeed essential and shared by all these processes. However, follow up studies to define the location of the ARPC1 protein and its existence within the ARP2/3 complex and/or association with actin filaments, will be required to fully solidify this conclusion. Figure 8. Actin cytoskeleton of root hairs, as visualized by expression of the 35S:GFP-ABD2GFP F-actin reporter in transgenic hairy roots. A, Wild type. B and C, Short root hair of arpc1. D, Longer root hair of arpc1. Scales in all pictures = 10 mm.
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MATERIALS AND METHODS Plant Material and Symbiotic Phenotypes Seed germination, plant growth conditions, and evaluation of infection and nodulation phenotypes were as described (Wopereis et al., 2000). For the assessment of mycorrhiza phenotypes, germinated seedlings were transferred to pots containing 1:1 mixture of Turface and sand soaked with one-half-strength Hoagland solution. Glomus intraradices-colonized leek (Allium porrum) roots were chopped into 1-cm pieces and used as the inoculum. The formation of AM symbiosis was evaluated at 6 and 8 weeks post inoculation with G. intraradices, following the previously described procedure (Kosuta et al., 2005).
Evaluation of Growth Parameters For analyses of root elongation and root hair phenotypes in the absence or presence of NF, plants were grown on the surface of vertically positioned agar plates as in Karas et al. (2005). NF isolation and root hair deformation assays were carried out as previously described (Miwa et al., 2006). Root hairs of soilgrown plants were evaluated and photographed 21 d after sowing. Shoot growth of uninoculated plants grown in the presence of the full Hoagland solution (Hoagland and Arnon, 1950) was measured 21 d after sowing using a metric ruler. At least 25 roots or shoots were examined and/or scored for each genotype in the above-described experiments. Pollen from wild-type and mutant flowers (approximately same stage) was germinated in vitro and evaluated as in Yokota et al. (2009).
Map-Based Cloning For map-based cloning of the ARPC1 locus, an F2 population was developed by crossing the arpc1 har1-1 (Gifu genetic background) mutant with the polymorphic Lotus japonicus MG-20_har1-1 introgression line (Murray et al., 2006). A total of 851 selected F2 arpc1 mutants were analyzed for cosegregation using available Simple Sequence Repeat, Cleaved/Cut Amplified Polymorphic Sequence (CAPS) and derived CAPS (dCAPS) markers (http:// www.kazusa.or.jp/lotus/). The arpc1 allele-specific SH1 MseI marker (Supplemental Table S1) was developed using dCAPS Finder 2.0 (http:// helix.wustl.edu/dcaps/dcaps.html).
ARPC1 Transcript Cloning The ARPC1 cDNA was amplified from nodule-derived total RNA using ThermoScript reverse transcription (RT)-PCR kit (Invitrogen) and oligo(dT)20 and the gene-specific primer (Supplemental Table S1). 59 and 39 RACE reactions were performed using the FirstChoice RLM-RACE kit (Ambion). For 59 and 39 RACE reactions the gene-specific primers ARPC159orR, ARPC159irR, ARPC139orF, and ARPC139irF, were used, respectively (Supplemental Table S2). The full-length ARPC1 cDNA, including the 59 and 39 untranslated regions, was amplified using the gene-specific primers ARPC1cDNA-F and ARPC1cDNA-R (Supplemental Table S2). The PCR reactions were carried out using the following conditions: 94°C (5 min), 30 cycles of 94°C (30 s), 58°C (30 s), 68°C (1.5 min), and 68°C (7 min) and high-fidelity platinum Taq DNA polymerase (Invitrogen). The resulting 59 and 39 RACE, and full-length cDNA products were cloned into pGEM-T easy vector (Promega) and were sequenced.
Expression Analysis Total RNA was extracted from various L. japonicus Gifu tissues using RNeasy plant mini kit (Qiagen, http://www.qiagen.com/), with the exception of shoots, cotyledons, flowers, and young green pods, where modified cetyl trimethyl ammonium bromide method was applied instead, as described (Kiefer et al., 2000). The synthesis of cDNA was performed using ThermoScript RT-PCR system (Invitrogen, www.invitrogen.com). The resulting cDNAs were subjected to real-time PCR using LightCycler 480 SYBR Green I master (Roche, www.roche-applied-science.com), following the manufacturer’s instructions. The ARPC1 gene-specific primers (ARPC1qRT-PCR-F and ARPC1qRT-PCR-R; Supplemental Table S2) were used in these experiments. The ubiquitin mRNA was amplified using UBC-F and UBC-R primer set (Supplemental Table S2) and served as an internal standard, as described (Tirichine et al., 2007). For each tissue under study, three biological and three technical replicates were used. All quantitative PCR reactions were performed 926
with the same cycling parameters of 95°C for 5 min, the 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. The amplification specificity was verified by analyzing the dissociation curve profile for each quantitative RT-PCR amplicon, and the efficiency of primers (Peff) was quantified using LinRegPCR (Ramakers et al., 2003). The mean (x–) and SD (s) of the cycle threshold (CT) value for each of the biological replicates was calculated for the target and reference genes using the three technical replicates. An average CT mean and SD encompassing the three biological replicates was then calculated. DCT values were calculated using the following formula: DCT = x– CT Target 2 x– CT Ubiquitin. DCT SD was calculated by: s DCT = [(s CT Target)2 2 (s CT Ubiquitin)2]1/2. The expression level (E) was calculated using the formula E = Peff(2DCt).
Complementation and Localization of the ARPC1 Gene Expression For in planta complementation experiments, the entire ARPC1 locus (13,638 bp) including 2.983 and 1.215 kb 59 and 39 untranslated regions, was amplified by PCR using Phusion enzyme (Finnzymes) and the gene-specific primers ARPC1genomic-F and ARPC1genomic-R (Supplemental Table S2). Fifty nanograms of the LjT48D13 large-insert plasmid DNA containing the ARPC1 locus was used as the template under the following cycling conditions: 98°C (30 s), 20 cycles of 98°C (10 s), 55°C (30 s), 72°C (6.5 min), and 72°C (10 min). The resulting PCR product was cloned into pDONR221 (Invitrogen) and subsequently recombined into the pKGWD,0 destination vector (Karimi et al., 2002), giving rise to the pKGWD,0-ARPC1 complementation construct. For the gene expression localization study, the ARPC1 promoter region (22983 to 295) was amplified by PCR using the ProARPC1-F and ProARPC1-R primer set (Supplemental Table S2) and the above-described PCR conditions. The resulting fragment was cloned into pENTR/D-TOPO Gateway vector (Invitrogen) and recombined into the pKGWFS7 destination vector (Karimi et al., 2002), giving rise to the ARPC1PRO:GUS/GFP expression construct. The integrity of all constructs was confirmed by sequencing. The vectors were introduced by Agrobacterium rhizogenes strain AR10-mediated transformation to generate transgenic hairy roots on nontransgenic wild-type and/or arpc1 mutant shoots, following the previously established procedure (Murray et al., 2007). For the genomic complementation and gene expression study, transgenic hairy roots were inoculated with wild-type Mesorhizobium loti strain NZP2235. The root hair phenotype of the resulting transgenic hairy roots was assessed while at the elongation stage in the liquid medium. Subsequently, the chimeric plants were planted in soil and inoculated with M. loti, and their symbiotic phenotypes were evaluated 21 d later. The transgenic hairy roots carrying the ARPC1PRO:GUS/GFP expression construct were collected at 14 and 21 dai. The actin structure was analyzed by imaging live root hairs in transgenic hairy roots expressing the 35S:GFP-ABD2-GFP F-actin reporter, as previously described (Yokota et al., 2009).
Microscopy Twenty-five to thirty-five micrometer sections of nodules were generated using agar-embedded explants and a vibrating-blade microtome (Leica), as previously described (Karas et al., 2005). Thin sectioning for light and transmission electron microscopies was performed as described (Madsen et al., 2010). For scanning electron microscopy of trichomes and cotyledons, tissues (young flower, leaves, and cotyledons) were fixed and dehydrated as described previously (Le et al., 2003). After critical point drying and coating, samples were observed using the Hitachi S-3400N scanning electron microscope. Images were processed using Adobe Photoshop CS3.
Computer Analyses Gene structure prediction was carried out by RiceGAAS (http://ricegaas. dna.affrc.go.jp/), GENSCAN (http://genes.mit.edu/GENSCAN.html), and National Center for Biotechnology Information Spidey mRNA to genomic alignment program (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/ Spidey/spideyweb.cgi). Analyses of protein structures and domain search were aided by SMART (http://smart.embl-heidelberg.de/) and PredictProtein (http://www.predictprotein.org/). Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession number JX446368. Plant Physiol. Vol. 160, 2012
ARPC1 Regulates Symbiotic Entry
Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Root hair deformation assay. Supplemental Figure S2. Map-based cloning. Supplemental Figure S3. Complementation of the arpc1 mutant phenotype. Supplemental Figure S4. Alignment of ARPC1 with known or predicted ARPC1 proteins from other organisms. Supplemental Table S1. Exon-intron structure of ARPC1 genes. Supplemental Table S2. Primers used in this study.
ACKNOWLEDGMENTS We thank A. Molnar for his expert help in preparation of figures. Received June 25, 2012; accepted August 1, 2012; published August 3, 2012.
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