RESEARCH ARTICLE

SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

Jing Liua,b, Miao Xia Liua, Li Ping Qiua and Fang Xiea,*

a National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China b University of the Chinese Academy of Sciences, Beijing, China * Correspondence Author: fxie@cemps.ac.cn

Short title: SPK1-ROP6 mediate polarized growth of ITs

One-sentence summary: SPIKE1, a DOCK family guanine nucleotide exchange factor, interacts with and activates the GTPase LjROP6 to mediate the polar progression of infection threads in Lotus japonicus.

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.plantcell.org) is: Fang Xie (fxie@cemps.ac.cn).

ABSTRACT In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.

INTRODUCTION 1  

To form symbiotic root nodules, legumes and rhizobia initiate their symbiotic 2  

interaction via a molecular dialog. The host legume secretes flavonoid compounds 3  

that function as signals sensed by rhizosphere rhizobia, and the expression of 4  

nodulation (Nod) genes is induced for the biosynthesis and secretion of Nodulation 5  

Factors (NF). NFs are lipochito-oligosaccharide molecular signals that are perceived 6  

Plant Cell Advance Publication. Published on October 6, 2020, doi:10.1105/tpc.20.00109

©2020 American Society of Plant Biologists. All Rights Reserved

by NF receptors. NFs initiate a series of plant signaling and developmental events, 7  

including root hair deformation, membrane depolarization, intracellular calcium 8  

oscillations, and cortex cell division. These events lead to the formation of the nodule 9  

primordium (Ehrhardt et al., 1992; Downie and Walker, 1999; Timmers et al., 1999; 10  

Cullimore et al., 2001; Esseling et al., 2003; Oldroyd and Downie, 2008). 11  

In temperate legumes such as Lotus japonicus and Medicago truncatula, rhizobia 12  

attach to the host’s root hairs tips and redirect root hair growth to entrap the rhizobia 13  

within an infection chamber (Esseling et al., 2003; Murray, 2011; Fournier et al., 14  

2015). Within the root hair chamber, an infection pocket filled with bacteria forms 15  

from a tubular invagination of the cell wall and membrane; this structure is known as 16  

an infection thread (IT). The IT extends from the infection chamber down through the 17  

root hairs to the root cortex, where a nodule primordium is produced (Fournier et al., 18  

2008; Fournier et al., 2015). The bacteria grow, divide, and fill the IT. The bacteria 19  

are then released from the IT into the nodule primordium via an unwalled droplet 20  

(Brewin, 2004). This leads to the formation of a nitrogen-fixing root nodule that 21  

provides the proper microenvironment for nitrogen fixation by rhizobia and nutrient 22  

exchange between the two partners (Jones et al., 2007; Oldroyd, 2013; Roy et al., 23  

2020). 24  

Genetic studies of L. japonicus and M. truncatula have revealed several genes 25  

that are required for IT formation, including LjNAP/MtRIT1, LjPIR, LjSCARN, and 26  

LjARPC1. These genes encode components of the WAVE/SCAR-ARP2/3 complex, 27  

which is required for actin nucleation and cytoskeletal rearrangement. LjNPL/MtNPL 28  

encode a legume-specific pectate lyase involved in cell wall remodeling. Mutant 29  

analyses revealed some genes that are required for rhizobial infection, but their 30  

precise biological/biochemical functions are unknown. These genes include 31  

LjRINRK1, encoding an atypical leucine-rich repeat receptor-like kinase; 32  

LjCERBERUS/MtLIN, encoding a U-box and WD40-repeat domain protein; 33  

MtVAPYRIN, encoding a VAP/MSP and ankyrin-repeats domain protein; MtRPG, 34  

encoding a coiled-coil protein; and MtCBS1, encoding a DUF21 and 35  

cystathionine-β-synthase domain protein (Yokota et al., 2009; Miyahara et al., 2010; 36  

Hossain et al., 2012; Qiu et al., 2015; Xie et al., 2012; Liu et al., 2019a; Li et al., 2019; 37  

Kiss et al., 2009; Yano et al., 2009; Murray et al., 2011; Arrighi et al., 2008, Sinharoy 38  

et al., 2016). 39  

The IT initiates and extends from the root hair tip to the nodule primordium. This 40  

process is directional, indicating that ITs show polarized growth. The polarized 41  

growth of pollen tubes and root hairs requires a strong calcium gradient at the tip, a 42  

polarized actin cytoskeleton, tip-directed vesicle trafficking, exocytosis, and signaling 43  

by ROP (Rho of plants) GTPases (Samaj et al., 2006; Kost, 2008; Craddock and Yang, 44  

2012; Craddock et al., 2012). The LIN-VAPYRIN-Exo70H4 protein complex in M. 45  

truncatula localizes to the tips of elongated pre-ITs, suggesting that an exocytosis 46  

process may be involved in the polarized growth of ITs (Liu et al., 2019b). Type I 47  

ROP GTPases (including ROP1 to ROP8) play important roles in root hair and pollen 48  

tube tip growth in Arabidopsis thaliana (Li et al., 1999; Kost et al., 1999; Jones et al., 49  

2002; Eklund et al., 2010; Craddock et al., 2012). The constitutively active (CA) 50  

forms of AtROP4 and AtROP6 do not function in the polarized growth of root hairs, 51  

nor do they form a localized Ca2+ gradient at the root hair tip (Molendijk et al., 2001). 52  

Compared to the wild type, plants expressing CA AtROP2 produce   additional and 53  

misplaced root hairs on the cell surface as well as longer root hairs, whereas plants 54  

expressing dominant negative (DN) forms of AtROP2 produce shortened wavy root 55  

hair (Jones et al., 2002). 56  

MtROP10 is associated with the NF receptor MtNFP and is involved in 57  

regulating the polarized growth of root hairs and NF-induced root hair deformation in 58  

M. truncatula (Lei et al., 2015). The roles of ROP genes in M. truncatula have been 59  

clarified to some extent by knock-down RNA interference (RNAi) studies. Compared 60  

to the wild type, MtROP8 RNAi transgenic plants show larger numbers of infection 61  

events and increased nodulation in response to inoculation, and MtROP9 RNAi plants 62  

show enhanced mycorrhizal and early hyphal root colonization but reduced rhizobial 63  

infection (Wang et al., 2014, Kiirika et al., 2012). In L. japonicus, the Nod factor 64  

receptor LjNFR5 interacts with LjROP6 to regulate IT formation and nodulation (Ke 65  

et al., 2012), and LjROP6 interacts with LjCHC1, a central component in 66  

clathrin-mediated endocytosis, to regulate rhizobial infection and nodule formation 67  

(Wang et al., 2015). These findings demonstrate that LjROP6 plays an essential role 68  

in regulating legume–rhizobium symbiosis. However, it is unknown whether ROP 69  

GTPase function in the polarized growth of ITs. 70  

ROP GTPases act as molecular switches in various signaling pathways by 71  

cycling between the GTP-bound active and GDP-bound inactive forms (Yang, 2002). 72  

The shuttling between the active and inactive forms of ROP GTPases is regulated by 73  

three types of factors: RhoGEF (guanine nucleotide exchange factor), which controls 74  

the transition of these GTPase from the inactive (GDP-bound) to active (GTP-bound) 75  

form; RhoGAP (GTPase-activating protein), which accelerates GTP hydrolysis; and 76  

RhoGDI (guanine nucleotide dissociation inhibitor), which inhibits GDP release so 77  

that ROP GTPases remain in their inactive state (Yang, 2002; Kost, 2008). The M. 78  

truncatula genome contains 10 genes encoding PRONE-type (plant Rop nucleotide 79  

exchanger) RopGEFs. MtRopGEF2 affects the cytosolic Ca2+ gradient and the 80  

subcellular architecture of root hairs and is required for root hair development (Riely 81  

et al., 2011). 82  

In addition to PRONE-type RopGEFs, a single DOCK family GEF, SPIKE1 83  

(SPK1), is also present in plants. In A. thaliana, spk1 seedlings show a lethal 84  

phenotype and display serious defects in the polarized growth of cotyledons and leaf 85  

epidermal cells (Qiu et al., 2002). The AtSPK1 DHR2 domain binds to ROPs to 86  

facilitate nucleotide exchange and generates signals to activate two heteromeric 87  

complexes, WAVE/SCAR and ARP2/3, to control actin polymerization (Basu et al., 88  

2008). AtSPK1 is involved in activating the AtROP6–AtRIC1 pathway, which 89  

regulates the auxin-mediated internalization of AtPIN2 (Lin et al., 2012). In   rice 90  

(Oryza sativa), OsSPK1 interacts with OsPit, an R protein against rice blast fungus, 91  

and regulates the activation of OsRac1 to trigger the immune response (Wang et al., 92  

2018). 93  

Although ITs show polar growth, it is unknown whether a ROP GTPase 94  

regulates this process and if so, how this ROP GTPase is activated. Here we show that 95  

LjSPK1 interacts with LjROP6 and activates its ROP GTPase activity. Some ITs were 96  

found to be misdirected in the root hairs of the Ljspk1 and Ljrop6 mutants after 97  

rhizobial infection. Our results suggest that an LjROP6-LjSPK1 signaling module 98  

guides the direction of IT growth during the early stage of rhizobial infection in L. 99  

japonicus. 100  

101  

RESULTS 102  

Expression Pattern of SPK1 in L. japonicus 103  

SPK1 is a DOCK family GEF that activates ROP GTPases (Basu et al., 2008). Using 104  

BLAST tools, we searched for the SPK1 gene in the L. japonicus genome. This search 105  

identified LjSPK1 (LotjaGi3g1v0299000), which encodes an 1829-amino acid protein 106  

showing 79.2% identity with AtSPK1. Phylogenetic analysis revealed that SPK1 is 107  

conserved across eukaryotes, usually as a single copy gene, with one exception being 108  

soybean (Glycine max), which has undergone a recent whole-genome duplication 109  

(Schmutz et al., 2010) (Supplemental Figure 1A). Sequence analyses revealed three 110  

conserved domains in LjSPK1: DHR1 (amino acids 446–726); DHR2 (amino acids 111  

1299–1829), which is the GEF catalytic domain; and DHR3 (amino acids 140–287) 112  

(Supplemental Figure 1B). To investigate the transcriptional pattern of LjSPK1 during 113  

nodulation, we monitored the transcript levels of LjSPK1 after NF treatment by 114  

RT-qPCR. LjSPK1 transcript levels slightly increased at 6 and 12 h after NF 115  

inoculation vs. the 0 h control (Figure 1A). 116  

We analyzed the spatial expression pattern of LjSPK1 by monitoring GUS 117  

expression from the GUS gene driven by the LjSPK1 promoter (pLjSPK1:GUS) in L. 118  

japonicus transgenic hairy roots. In the absence of rhizobial inoculation, we detected 119  

GUS signals in root vascular tissues and lateral roots (Figure 1B and C). However, 120  

after inoculation with Mesorhizobium loti R7A/lacZ, GUS signals were present in the 121  

dividing cortex region (Figure 1D), nodule primordia (Figure 1E), bumps (Figure 1F), 122  

young nodules (Figure 1G), and vascular bundles of mature nodules (Figure 1H). 123  

Light microscopy analyses of sections of these nodules revealed GUS signals in bump 124  

and young nodule cells (Figure 1I and J), but not in the nitrogen-fixation zone of 125  

mature nodules (Figure 1K). These expression patterns suggest that LjSPK1 might be 126  

involved in root nodule symbiosis in L. japonicus. 127  

128  

LjSPK1 Is Required for Polarized Growth in Plants and Rhizobial Infection 129  

To explore the roles of LjSPK1 in root nodule symbiosis, we obtained two LORE1 130  

insertion lines (30019873 and 30051253) in which LjSPK1 had insertions at amino 131  

acid positions 273 and 910 from the start codon, respectively. These lines were 132  

designated as Ljspk1-1 (30019873) and Ljspk1-2 (30051253) (Supplemental Figure 133  

1B). RT-qPCR analysis revealed that endogenous LjSPK1 transcript levels were 134  

significantly reduced in both Ljspk1 lines (Supplemental Figure 1C). For both Ljspk1 135  

alleles, the homozygotes were nearly completely sterile, so we could only use 136  

seedlings obtained from the segregation of heterozygous plants for phenotypic 137  

analyses. Soil-grown Ljspk1 mutants survived but showed some growth defects, 138  

including severe dwarfism of adult plants (Supplemental Figure 1D), few and smaller 139  

flowers with an abnormal petal shape (Supplemental Figure 1E), and smaller, 140  

dark-green leaves (Supplemental Figure 1F–H). Wild-type plants formed elongated, 141  

filamentous trichomes on the abaxial midribs of leaves, whereas the Ljspk1 mutants 142  

formed fewer and smaller trichomes (Supplemental Figure 1H). Scanning electron 143  

microscopy of epidermal cells on the dorsal leaves of wild type (Gifu) and Ljspk1 144  

mutants revealed that wild-type pavement cells had a clear neck and lobe, whereas 145  

those on Ljspk1 leaves were nearly round (Supplemental Figure 1I and J). The 146  

phenotypes of these Ljspk1 mutants were similar to those reported for the Arabidopsis 147  

spk1-1 mutant (Qiu et al., 2002). These results suggest that LjSPK1 is involved in the 148  

polarized growth of cells in L. japonicus. 149  

We analyzed the infection and nodulation phenotypes of the Ljspk1 mutants 150  

following inoculation with M. loti R7A constitutively expressing GFP or lacZ. Wild 151  

type (Gifu) and Ljspk1 wild-type siblings (sibling) produced normal elongated ITs in 152  

curled root hairs (Figure 2A, Supplemental Figure 2A, B and D). In the Ljspk1 153  

mutants, infection initiated normally from a curled root hair, but some ITs showed 154  

anomalous   growth patterns, including the formation of sac-like structures or 155  

depolarized loop shapes inside the root hairs (Figure 2B–D, Supplemental Figure 2C 156  

and E).  We counted the total number of infection events in the Ljspk1 mutants 1 week 157  

after inoculation. The number of infection events including infection foci (foci), ITs in 158  

root hairs (IT), and ITs extending into cortex cells (rIT) was significantly lower in the 159  

Ljspk1 mutants than in wild type. In addition, there were significantly more abnormal 160  

ITs (“abnormal” in Figure 2) in the Ljspk1 mutants than in wild type (Figure 2E). 161  

Because the Ljspk1 mutants had shorter primary roots than the wild type 162  

(Supplemental Figure 2F and I), we calculated the number of infection events per 163  

centimeter (cm). The number of infection foci and ITs per cm root did not differ 164  

significantly between wild type and the Ljspk1 mutants except that Ljspk1-2 had 165  

fewer rITs (Figure 2F). However, both Ljspk1 mutants had more abnormal infection 166  

events than the wild type and Ljspk1 siblings (Figure 2F). These results suggest that 167  

the infection events were not significantly affected, but the polarized growth of ITs 168  

was markedly affected, in the Ljspk1 mutants. At 2 weeks after inoculation, the wild 169  

type had produced abundant pink mature nodules, while the Ljspk1 mutants had 170  

produced fewer nodules per plant (Supplemental Figure 2G, J and K). Light 171  

microscopy of semi-thin sections of nodules stained with toluidine-blue revealed that 172  

Ljspk1-1 nodules had fewer infected cells than the wild type and Ljspk1-1 siblings’ 173  

nodules (Supplemental Figure 2H). 174  

Next, we explored the role of LjSPK1 by generating LjSPK1-overexpressing 175  

lines (LjSPK1-OX) and lines with knocked-down LjSPK1 expression (LjSPK1-Ri). 176  

The LjSPK1-knockdown lines were generated by introducing an RNAi vector and the 177  

LjSPK1-OX lines were generated by introducing the LjSPK1 cDNA driven by the L. 178  

japonicus Ubiquitin promoter into wild-type L. japonicus hairy roots. Compared with 179  

empty vector (EV) control, the LjSPK1-Ri lines had markedly fewer infection events, 180  

but the LjSPK1-OX lines had increased numbers of infection events, including 181  

infection foci and ITs (Figure 2G and H). The LjSPK1-Ri lines had shorter roots than 182  

the EV control (Supplemental Figure 2L). Analysis of the infection events revealed 183  

that both the LjSPK1-Ri and LjSPK1-OX lines had more abnormal infection events per 184  

cm of root than the EV control (Figure 2I). The phenotype of LjSPK1-Ri was similar 185  

to that of the Ljspk1 LORE1 insertion mutants. RT-qPCR analysis revealed that 186  

LjSPK1 transcript levels were significantly lower in the LjSPK1-Ri lines and 187  

significantly higher in the LjSPK1-OX lines compared to the control (Supplemental 188  

Figure 2M). Together, these results demonstrate that LjSPK1 is required for the 189  

rhizobial infection process and is involved in the polarized growth of ITs in L. 190  

japonicus. 191  

192  

SPK1 Interacts with Three L. japonicus ROP GTPases in Yeast Cells, but Only 193  

LjROP6 Is Required for Polarized Growth of ITs 194  

AtSPK1 is a DOCK-family GEF protein, and DHR2 is the GEF catalytic domain that 195  

facilitates the nucleotide exchange activity of ROP GTPases (Basu et al., 2008). We 196  

were interested in determining which ROP GTPase is activated by LjSPK1 to regulate 197  

rhizobial infection in L. japonicus. BLAST searches of the L. japonicus genome 198  

revealed 10 ROP GTPases. We conducted phylogenetic analysis of ROP GTPases in 199  

A. thaliana, L. japonicus, and M. truncatula (Supplemental Figure 3A). Because 200  

AtSPK1 interacts and facilitate nucleotide exchange with type I ROP GTPase, and 201  

most ROP GTPases are membrane-localized proteins, we used the DUAL membrane 202  

yeast two-hybrid system to determine which type I ROP GTPase interacts with the 203  

LjSPK1 DHR2 domain (named SPK1-DHR2). Of the four type I and one type II ROP 204  

GTPases examined, three type I ROP GTPases (LjROP1, LjROP3, and LjROP6) 205  

interacted with the LjSPK1 DHR2 domain in yeast cells (Supplemental Figure 3B). 206  

No interaction was detected between LjSPK1 DHR2 and LjROP5 or LjROP10 in 207  

yeast cells (Supplemental Figure 3B). 208  

To explore whether these three type I LjROPs function in rhizobial infection, we 209  

obtained their LORE1 insertion mutants (30000786   with an insertion at 83 bp in 210  

LjROP1; 30000537 with an insertion at 67 bp before the start codon in LjROP3; and 211  

30031226 with an insertion at 258 bp in LjROP6). We then obtained homozygotes of 212  

these insertion mutants. RT-qPCR analysis confirmed that endogenous LjROP 213  

transcript levels were significantly reduced in all three Ljrop mutants (Supplemental 214  

Figure 3C). We analyzed the root hair and infection phenotypes of the mutants. First, 215  

we observed the root hairs of the mutants at 2 days after germination with or without 216  

rhizobial inoculation. In the absence of rhizobial inoculation, the roots of wild-type 217  

plants formed straight root hairs, but more than half of the plants in lines Ljrop1 218  

(15/30), Ljrop3 (18/26), and Ljrop6-1 (20/34) formed branched root hairs or root hairs 219  

with swollen tips (Figure 3A). At 18 h after M. loti R7A/lacZ inoculation, both the 220  

wild type and Ljrop mutants displayed rhizobia-induced root hair deformation in the 221  

infection zone, with no distinguishable differences between these lines (Figure 3B). 222  

These observations suggest that all three LjROP GTPases play roles in root hair 223  

development and respond normally to rhizobia. 224  

We further analyzed IT formation and infection events after rhizobial inoculation. 225  

There were no differences in IT formation or infection events between wild type and 226  

Ljrop1 or Ljrop3, but there were fewer infection events in Ljrop6-1 than in wild type 227  

(Figure 3G). Interestingly, Ljrop6-1 contained some misdirected ITs in the root hairs, 228  

which were similar to, but more severe than, those in Ljspk1 (Figure 3F and described 229  

in detail below). However, this phenotype was not observed in wild type, Ljrop1, or 230  

Ljrop3 plants (Figure 3C–E). To analyze whether these LjROPs play functionally 231  

redundant roles during rhizobial infection, we crossed Ljrop1 × Ljrop3 and Ljrop1 × 232  

Ljrop6-1 to obtain the Ljrop1 Ljrop3 and Ljrop1 Ljrop6-1 double mutants. We 233  

analyzed the infection events in these mutants after inoculation with M. loti 234  

R7A/LacZ. Compared with wild-type plants, Ljrop1 Ljrop3 plants had more infection 235  

foci but no misdirected ITs, and Ljrop1 Ljrop6-1 plants produced approximately the 236  

same number of misdirected ITs as Ljrop6-1 (Supplemental Figure 3D). These results 237  

indicate that ROP1, ROP3, and ROP6 affect the polarized growth of root hairs in L. 238  

japonicus, but only LjROP6 is required for the polarized growth of ITs in root hairs. 239  

The results also indicate that the misdirection of ITs in Ljrop6-1 is not caused by a 240  

deficiency in root hair development. 241  

242  

LjROP6 Is Required for the Polarized Growth of ITs in L. japonicus 243  

To confirm the notion that ROP6 is required for the polarized growth of ITs in root 244  

hairs, we obtained two more LORE1 insertion lines: 30142846 (Ljrop6-2) and 245  

30103232 (Ljrop6-3), with insertions at 505 bp after and 114 bp before the LjROP6 246  

start codon, respectively (Supplemental Figure 4A). RT-qPCR analysis of 247  

homozygous Ljrop6-2 and Ljrop6-3 plants showed that LjROP6 transcript levels were 248  

significantly lower in these plants than in wild-type plants (Supplemental Figure 4E). 249  

We observed the root hair phenotypes of the Ljrop6-2 and Ljrop6-3 mutants at 2 250  

days after germination. Both before and after rhizobial inoculation, the root hair 251  

phenotypes and responses to rhizobia of the Ljrop6-2 and Ljrop6-3 mutants were 252  

similar to those of Ljrop6-1 (Supplemental Figure 5A and B). We analyzed rhizobial 253  

symbiosis in these three Ljrop6 mutants after M. loti R7A/LacZ inoculation. After 254  

rhizobial infection, the three Ljrop6 mutants produced normal ITs like those in the 255  

wild type (Figure 4A). However, as noted above, some ITs in the Ljrop6 mutants 256  

were misdirected or tangled in the root epidermal cells (Figure 4B–E), suggesting that 257  

the ITs became misdirected as they elongated from the infection chamber down to the 258  

cortex nodule primordium. Compared to the wild type, the Ljrop6 mutants had 259  

significantly fewer infection events and formed more misdirected ITs at 5 days after 260  

inoculation with M. loti R7A/lacZ (Figure 4F and G). At 2 weeks after M. loti 261  

R7A/lacZ inoculation, the Ljrop6 mutants and wild-type plants produced normal 262  

mature pink nodules (Supplemental Figure 4B). The total nodule number was not 263  

significantly different between the Ljrop6 mutants and wild-type plants 264  

(Supplemental Figure 4C and D). 265  

To further confirm the notion that LjROP6 is required for the polarized growth 266  

of ITs, we generated transgenic hairy roots in the Ljrop6-1 background using the 267  

LjROP6 native promoter to drive the expression of LjROP6 cDNA fused with 268  

mCherry (pLjROP6:LjROP6-mCherry). The disorientated ITs in Ljrop6-1 were 269  

rescued by complementation with ROP6-mCherry (Figure 4H). Statistical analyses 270  

showed that the complemented group (pLjROP6:LjROP6-mCherry/Ljrop6-1) had 271  

more ITs and rITs and fewer misdirected ITs compared with the EV control at the 272  

same time point (Figure 4I). LjROP6 transcript levels were significantly higher in 273  

Ljrop6-1 hairy roots than the control (Supplemental Figure 4F). Together, these 274  

observations confirm the notion that LjROP6 is required for the polarized growth of 275  

ITs in L. japonicus. 276  

277  

LjSPK1 Physically Interacts with LjROP6 in Planta and Activates Its GTPase 278  

Activity in Vitro 279  

As described above, LjROP6 interacted with LjSPK1 in yeast cells. We further 280  

confirmed their interaction in Nicotiana benthamiana leaf pavement cells using 281  

split-luciferase complementation and co-immunoprecipitation (Co-IP) assays. 282  

LjROP6 interacted with the LjSPK1-DHR2 catalytic domain in split-luciferase 283  

complementation (Figure 5A) and Co-IP assays (Figure 5B). However, we did not 284  

detect co-precipitation of LjSPK1 with LjROP10, a type II ROP GTPase, in our Co-IP 285  

assays (Figure 5B). The activation of ROP GTPases relies on GDP and GTP exchange, 286  

and this cycle requires GEFs to facilitate the dissociation of GDP (Kost, 2008). We 287  

therefore investigated the ability of LjSPK1 to interact with LjROP6 in the dominant 288  

negative (DN, D121A) or constitutively active (CA, G15V) form. LjSPK1-DHR2 289  

interacted more strongly with the LjROP6 DN form than with the LjROP6 CA form 290  

in Co-IP assays (Figure 5B). These results suggest that LjSPK1 preferentially 291  

interacts with the inactive form, but not the active form, of LjROP6 to activate the 292  

nucleotide exchange activity of LjROP6. 293  

The LjSPK1 DHR2 domain is known to activate small GTPases (Basu et al. 294  

2008, Wang et al. 2018). To examine whether LjSPK1 facilitates the guanine 295  

nucleotide exchange activity of LjROP6, we tested the GEF activity of the LjSPK1 296  

DHR2 domain in vitro using a fluorescence spectroscopy-based procedure (Gu et al., 297  

2006, Wang et al., 2017). First, we expressed and purified GST-tagged 298  

LjSPK1-DHR2 and unlabeled-GDP His-tagged LjROP6 in Escherichia coli. To start 299  

the nucleotide exchange reaction, we added fluorescently labeled 300  

N-methylanthraniloyl (mant)-GTP and unlabeled-GDP LjROP6 to the reaction buffer 301  

and recorded the fluorescence values for 600 s. The intrinsic guanine nucleotide 302  

exchange rate of LjROP6 increased with increasing reaction time and with increasing 303  

concentrations of LjROP6 (Figure 5C). Next, to investigate the effect of LjSPK1 on 304  

the guanine nucleotide exchange rate of LjROP6, we added LjSPK1-DHR2 protein to 305  

reaction buffer containing 0.5 µM unlabeled-GDP LjROP6 protein and 1 µM 306  

mant-GTP before recording the change in fluorescence intensity. The LjSPK1 GEF 307  

activity towards LjROP6 increased with increasing LjSPK1-DHR2 concentrations 308  

(Figure 5D). These results demonstrate that LjROP6 has intrinsic guanine nucleotide 309  

exchange activity, which is enhanced by SPK1. 310  

311  

LjROP6 Co-localizes and Interacts with LjSPK1 in the Plasma Membrane 312  

LjROP6 was shown to be localized to the plasma membrane (PM) and cytoplasm (Ke 313  

et al., 2012), but SPK1 is localized to the endoplasmic reticulum (ER) (Zhang et al., 314  

2010; Wang et al., 2018). To validate the subcellular localization of LjROP6 and 315  

confirm its interaction with LjSPK1, we analyzed the subcellular localization of 316  

LjROP6 in N. benthamiana leaf cells and L. japonicus roots. We co-expressed 317  

GFP-LjROP6 or LjROP6-mCherry under the control of the L. japonicus Ubiquitin 318  

promoter in N. benthamiana leaf cells. In these analyses, LjROP6 localized to the PM, 319  

regardless of whether it was tagged with a fluorescent marker at its N-terminus or 320  

C-terminus (Supplemental Figure 6A–C). We then expressed LjROP6-mCherry in 321  

wild-type L. japonicus hairy roots and monitored its localization before and after 322  

inoculation with M. loti R7A/mTag. Signals from mCherry were observed at the PM 323  

in root hairs without rhizobia (Supplemental Figure 6D) and in elongating or curled 324  

root hairs after inoculation with M. loti R7A (Supplemental Figure 6E). The 325  

LjROP6-mCherry fusion protein displayed a similar subcellular localization pattern 326  

regardless of whether it was expressed under the control of the Ubiquitin promoter 327  

(Supplemental Figure 6D and E) or the LjROP6 native promoter (Supplemental 328  

Figure 6F). 329  

Next, we analyzed the subcellular localization of LjSPK1. We transformed N. 330  

benthamiana leaves with Agrobacterium carrying LjSPK1-YFP and observed the 331  

localization of LjSPK1 by monitoring the fluorescence of YFP by laser scanning 332  

confocal microscopy. LjSPK1 showed a punctate distribution close to the PM or 333  

cytoplasm (Figure 6A). Following plasmolysis using 30% sucrose, LjSPK1-YFP was 334  

not associated with the cell wall (Figure 6B). To examine whether LjSPK1 localizes 335  

to the PM, we stained the N. benthamiana leaves with the   PM localization marker 336  

FM4-64. LjSPK1 showed almost no co-localization with FM4-64 (Figure 6C and E). 337  

AtSPK1 and OsSPK1 are localized to subdomains of the ER (Zhang et al., 2010, 338  

Wang et al., 2018). To determine whether this is also the case for LjSPK1, we 339  

co-expressed LjSPK1-YFP with the ER marker HDEL-mRFP in N. benthamiana 340  

leaves. LjSPK1 colocalized with the ER marker (Figure 6D and F). These findings 341  

suggest that LjSPK1 is associated with subdomains of the ER. We then explored the 342  

subcellular distribution of LjSPK1-eGFP in L. japonicus hairy roots. LjSPK1-eGFP 343  

displayed PM and punctate distribution close to the nucleus in L. japonicus root hairs 344  

(Figure 6G), and LjSPK1-eGFP also co-localized with the   PM marker FM4-64 345  

(Figure 6H). LjSPK1-GFP expressed in Ljrop6-1 hairy roots also showed a punctate 346  

distribution close to the nucleus (Supplemental Figure 7A), similar to that of 347  

LjSPK1-eGFP expressed in the wild type. After rhizobia inoculation, LjSPK1-eGFP 348  

was detected in the PM of root hairs with infection foci or elongated ITs (Figure 6I 349  

and J). 350  

We then co-expressed LjSPK1-YFP and LjROP6-mCherry in N. benthamiana 351  

leaf pavement cells. In contrast to the punctate localization of LjSPK1-YFP when 352  

expressed alone, LjSPK1-YFP co-expressed with LjROP6-mCherry showed reduced 353  

punctate distribution, and most LjSPK1-YFP signals were localized in patches on the 354  

PM (Figure 7A–C). This observation suggests that LjROP6 promotes the PM 355  

localization of LjSPK1. 356  

We evaluated the interaction between LjSPK1 and LjROP6 in planta via 357  

bimolecular fluorescence complementation (BiFC) assays, with C-terminal and 358  

N-terminal split-Venus fragments fused to LjSPK1 and LjROP6, respectively, driven 359  

by the L. japonicus or A. thaliana Ubiquitin promoter. When this construct 360  

(pLjUb:LjSPK1-cVenus/pAtUb:LjROP6-nVenus) was agro-infiltrated into N. 361  

benthamiana leaf cells, Venus fluorescence was detected in the PM and in some 362  

punctate structures adjacent to the PM (Figure 7D). This distribution was confirmed 363  

by plasmolysis experiments using 30% sucrose (Figure 7E). These results confirm the 364  

notion that LjSPK1 interacts with LjROP6 at the PM. We then introduced this 365  

construct into wild-type L. japonicus hairy roots and monitored the localization of 366  

Venus fluorescence signals after inoculation with M. loti R7A/mTag. Venus 367  

fluorescence signals were detected in the PM of deformed root hairs (Figure 7F) as 368  

well as in infection foci (Supplemental Figure 7B). We also expressed these 369  

constructs in the M. truncatula sunn-1 mutant, which had many more infection events 370  

than wild type. Venus fluorescence was observed in the infection chamber of the root 371  

hairs at the site of the initiating IT (Figure 7G and Supplemental Figure 7C). 372  

373  

Polarized Growth of Root Hairs and Rhizobial Infection Are Affected in 374  

LjROP6-overexpression, CA, and DN Lines 375  

To explore the   role of LjROP6 in root hair development and IT formation, we 376  

generated LjROP6-overexpression, LjROP6 CA, and LjROP6 DN hairy roots with 377  

each construct under the control of the L. japonicus Ubiquitin promoter. Green 378  

fluorescent protein (GFP) signals were visualized in the transformed roots. In the EV 379  

control, root hairs showed normal tip growth, and the root hairs grew straight and 380  

away from the primary root axis (n=30) (Supplemental Figure 8A and B). However, 381  

in hairy roots overexpressing LjROP6, root hairs were morphologically similar to 382  

those of the control in that they displayed normal tip growth, but approximately 383  

one-third of the hairy roots formed shorter root hairs (25/82) (Supplemental Figure 384  

8A–C). Surprisingly, all LjROP6 CA (42/42) and LjROP6 DN (51/51)   lines formed 385  

completely depolarized ballooning root hairs on transgenic roots (Supplemental 386  

Figure 8A–C). 387  

We analyzed the infection events in the LjROP6-overexpression, LjROP6 CA, 388  

and LjROP6 DN transgenic hairy roots after M. loti R7A/lacZ inoculation. The 389  

LjROP6-overexpression line formed elongated ITs similar to those of the EV control 390  

but had a few infection foci in uncurled root hairs and formed some disorientated ITs 391  

(Supplemental Figure 8D and E). The number of infection events (foci and ITs) in 392  

hairy roots appeared to be slightly higher in the LjROP6-overexpression line than in 393  

the EV control, but this difference was not significant (Supplemental Figure 8H). In 394  

the hairy roots of LjROP6 CA and LjROP6 DN, most ITs were abnormally short and 395  

formed without root hair curling (Supplemental Figure 8F and G). In addition, the 396  

number of infection events was significantly reduced in LjROP6 CA and LjROP6 DN 397  

hairy roots vs. the EV control (Supplemental Figure 8H). LjROP6 transcript levels in 398  

hairy roots were higher in the LjROP6-overexpression, LjROP6 CA, and LjROP6 DN 399  

lines than in the EV control (Supplemental Figure 8I). These observations demonstrate 400  

that LjROP6 homeostasis is essential for its function in root hair development and IT 401  

formation. 402  

We investigated the functions of LjROP1 and LjROP3 in root hair development by 403  

expressing LjROP1 or LjROP3 cDNA under the control of the LjUb promoter in 404  

wild-type L. japonicus hairy roots and analyzing their CA and DN forms. In all of 405  

these transgenic lines, the hairy roots formed shorter root hairs than the EV control 406  

(Supplemental Figure 9), which is similar to the phenotype of LjROP6-overexpressing 407  

L. japonicus hairy roots. 408  

409  

Actin filaments are Disordered in the Root Hairs of Ljrop6-1 Compared to 410  

Wild-type L. japonicus 411  

The actin cytoskeleton is important for root hair tip growth. To determine whether 412  

LjROP6 regulates the arrangement of actin filaments to influence the polarized 413  

growth of root hair tips, we used Alexa Fluor 488-conjugated phalloidin to stain wild 414  

type and Ljrop6-1 roots. As expected, the root hairs of wild-type plants predominantly 415  

displayed the characteristic arrangement of actin filaments in long cables aligned 416  

longitudinally (Supplemental Figure 10A). However, 70% of Ljrop6-1 root hairs had 417  

fewer longitudinally aligned actin filaments and significantly more transversely 418  

oriented ones compared to the wild type (Supplemental Figure 10B). Most of the short 419  

swollen or medium-length root hairs of Ljrop6-1 showed disordered or web-like 420  

arrangements of actin filaments (Supplemental Figure 10C). These results indicate 421  

that LjROP6 affects the arrangement of actin filaments in root hairs to regulate their 422  

development. 423  

424  

DISCUSSION 425  

Rhizobial ITs elongate following the migration of the nucleus along and within the 426  

root hairs via polar tip growth. ROP GTPases are key components required for the 427  

polarized growth of cells during processes such as pollen tube elongation and root 428  

hair development (Zheng and Yang, 2000; Samaj et al., 2006). LjROP6 interacts with 429  

the NF receptor LjNFR5 to mediate nodulation, and LjROP6 associates with LjCHC1 430  

(Ke et al., 2012; Wang et al., 2015). The CHC1-hub domain, a dominant effector of 431  

clathrin-mediated endocytosis, impairs rhizobial infection (Wang et al., 2015). Here, 432  

we demonstrated that LjROP6 is required for the polarized growth of ITs in root hairs. 433  

GTPases act as molecular switches that fluctuate between an inactive GDP-bound 434  

form and an active GTP-bound form. We found that the DOCK-family GEF protein 435  

LjSPK1 interacts with, and activates, LjROP6. We also found that LjROP6 promotes 436  

the localization of LjSPK1 to the PM and that this may be important for its function. 437  

Together, our results show that the DOCK-family GEF LjSPK1 activates LjROP6 to 438  

mediate the polarized growth of ITs, and they provide evidence that early NF 439  

signaling is connected to morphological changes associated with rhizobial infection. 440  

Rhizobial invasion into host plants via the IT requires an actively growing root 441  

hair. Inside the root hair, an infection chamber forms and elongates to form the IT, the 442  

tubular structure that guides bacteria toward cortical cells (Fournier et al., 2008, 443  

Fournier et al., 2015). ROP GTPases play important roles in the polarized growth of 444  

cells. ROPs are polarized into single, discrete PM domains in tip-growing cells such 445  

as pollen tubes and root hairs (Lin et al., 1996; Molendijk et al., 2001). The NF 446  

receptor LjNFR5 interacts with LjROP6 to mediate IT formation and nodulation (Ke 447  

et al., 2012). In the current study, we identified three LORE1 insertion mutants of 448  

Ljrop6. All three mutants were able to form normal ITs after rhizobial inoculation. 449  

However, compared to the wild type, the mutants had fewer infection events (foci and 450  

ITs) and formed many more disoriented ITs in root hairs. This observation suggests 451  

that LjROP6 mediates the polarized growth of ITs in root hairs. 452  

Disoriented ITs have also been observed in other mutants, such as mutants of the 453  

cytokine receptor gene LjLhk1 (Murray et al., 2007) and topoisomerase VI (TOPO6A) 454  

subunit LjSUNERGOS1 or LjVAG1 (Suzaki et al., 2014, Yoon et al., 2014). These 455  

mutants exhibit misdirected ITs and highly defective nodule organogenesis, which in 456  

turn affects nodule primordium formation. However, in the Ljrop6 mutants observed 457  

in the current study, the ITs formed loops and showed disoriented growth in root 458  

epidermis cells, but nodule formation  was not affected. This finding suggests that the 459  

LjROP6-mediated polar elongation of ITs is not associated with nodule primordium 460  

formation. The Golgi/trans-Golgi-localized Rab GTPase PvRabA2 is required for IT 461  

progression and the maintenance of membrane integrity in common bean (Phaseolus 462  

vulgaris) (Blanco et al., 2009; Dalla et al., 2017). In M. truncatula, the 463  

LIN-VPY-Exo70H4 complex shows a polar distribution in pre-ITs, suggesting that 464  

this complex mediates the polarized growth of ITs via exocytosis (Liu et al., 2019b). 465  

In the future, it would be interesting to determine whether LjROP6 interacts with this 466  

complex to guide the polarized growth of ITs. 467  

ROP GTPases regulate cell growth and shape by organizing the actin filaments 468  

that drive cytoplasmic streaming and vesicle trafficking in the cytoskeleton. These 469  

GTPases also affect the arrangement of cortical membrane-associated microtubules, 470  

which guide cell wall deposition via cellulose synthase complexes (Fu et al., 2005; 471  

Endler and Persson, 2011; Tominaga and Ito, 2015). ROP GTPase activity is 472  

regulated by GAPs, GEFs, and RhoGDIs (Yang, 2002; Kost, 2008). SPK1 is a 473  

DOCK-family GEF that is conserved in animals and plants (Meller et al., 2005). In 474  

Arabidopsis, AtSPK1 has been implicated in activating ROP signaling to regulate 475  

actin polymerization via WAVE and ARP2/3 complexes in leaves (Qiu et al., 2002; 476  

Basu et al., 2008). Similarly, in rice, OsPit interacts with OsSPK1 to activate 477  

OsRAC1 and trigger immune responses (Wang et al., 2019). The current results 478  

indicate that LjSPK1 interacts with and activates LjROP6 and that both LjSPK1 and 479  

LjROP6 are involved in the polarized growth of ITs following rhizobial infection. The 480  

formation of ITs requires several components of the WAVE/SCAR-ARP2/3 complex, 481  

including NAP, PIR, SCARN, and ARPC1 (Yokota et al., 2009; Hossain et al., 2012; 482  

Qiu et al., 2015). It will be important to investigate whether the LjSPK1-LjROP6 483  

module interacts with the WAVE/SCAR-ARP2/3 complex to regulate actin 484  

rearrangement and mediate IT formation. 485  

Exocytosis and the polar inhibition of clathrin-dependent endocytosis regulate 486  

the polarized growth of cells in both animals and plants. Studies of ROPs/RACs have 487  

revealed that these processes are important targets of Rho-GTPase signaling 488  

(Craddock et al., 2012; Craddock and Yang, 2012; Feiguelman et al., 2018). For 489  

example, the yeast exocyst subunit SEC3 is a direct effector of the Rho GTPase 490  

Cdc42. In Arabidopsis, the ROP effector ICR1/RIP1 (INTERACTOR OF 491  

CONSTITUTIVE ACTIVE ROP1) interacts with the Arabidopsis SEC3 homolog and 492  

is required for the polar localization of PIN1 (Lavy et al., 2007; Hazak et al., 2010). 493  

SPK1 is required for the auxin-activated ROP6-RIC1 pathway to inhibit the 494  

internalization of PIN2 by stabilizing actin microfilaments. These processes affect 495  

auxin distribution and lateral root development (Lin et al., 2012). In L. japonicus, 496  

LjROP6 interacts with LjCHC1 and is involved in rhizobial infection (Wang et al., 497  

2015). Interestingly, LjROP6 alone localizes to the PM but shows a punctate 498  

distribution on the PM when it is co-expressed with LjCHC1 (Wang et al., 2015) or 499  

LjSPK1, as demonstrated in the BiFC assay between LjSPK1 and LjROP6 in N. 500  

benthamiana leaves in the current study. Further studies are needed to investigate 501  

whether LjSPK1-activated LjROP6 can activate other downstream effectors such as 502  

RICs or RIPs. Such interactions could control the polarized growth of rhizobial ITs 503  

via effects on the organization of actin microfilaments, auxin distribution, and 504  

exocytosis and endocytosis pathways. 505  

AtSPK1 is localized to subdomains of the ER (Zhang et al., 2010), and OsSPK1 506  

(Wang et al., 2018) and LjSPK1 (this study) show a punctate distribution and 507  

co-localize with an ER marker. However, co-expressing OsSPK1-OsRAC1 or 508  

LjSPK1-LjROP6 in N. benthamiana leaves enhanced the PM localization of SPK1, 509  

suggesting that OsSPK1-OsRAC1 or LjSPK1-LjROP6 interact at the PM (Wang et al., 510  

2018). In the current study, LjSPK1 alone was distributed at the PM and in punctate 511  

spots around the nucleus in L. japonicus, but it interacted with LjROP6 in the PM and 512  

infection pocket in curled root hairs of L. japonicus infected with M. loti. In rice, 513  

OsRacGEF1 is transported from the ER to the PM with OsCERK1 via a vesicle 514  

trafficking pathway (Akamatsu et al., 2013). It would be interesting to explore which 515  

pathway is involved in the translocation of LjSPK1 from the ER to the PM. 516  

GEFs are activated by PM-bound receptor-like kinases (RLKs) (Akamatsu et al., 517  

2013; Liao et al., 2017). In mammals, the kinase Akt and the protein phosphatase 518  

PP2A interact with the DHR2 domain of DOCK6. DOCK6 is phosphorylated by Akt 519  

and dephosphorylated by PP2A at Ser1194; the phosphorylation status of DOCK6 520  

determines its ability to promote axon growth (Miyamoto et al., 2013). A 521  

phosphoproteomic study of root nodule symbiosis in M. truncatula revealed that two 522  

phosphoisoforms of DOCK-family proteins, MtSPK1 and DOCK7, displayed lower 523  

phosphorylation levels upon NF treatment in nfp and dmi3 than in the wild type. 524  

Those findings suggest that rhizobia infection may affect the phosphorylation status 525  

of MtSPK1 (Rose et al., 2012). Thus, it will be important to investigate whether there 526  

is a kinase that affects the phosphorylation status of LjSPK1 to regulate its activity 527  

towards LjROP6 during the establishment of symbiosis. 528  

ROP GTPases play an essential role in controlling the polarized growth of pollen 529  

tubes and root hairs. In Arabidopsis, AtSPK1 functions as a GEF that interacts with 530  

and activates a series of ROP GTPases (Basu et al., 2008). Our results show that 531  

LjSPK1 interacts with three type I ROP GTPases in L. japonicus. All three LjROP 532  

GTPases are required for the polarized growth of root hairs. The Ljrop mutants in this 533  

study formed branched and swollen root hairs, while lines overexpressing the LjROP 534  

GTPases and their CA or DN forms produced short, ballooning root hairs. These 535  

phenomena are not consistent with the previous finding that plants expressing the DN 536  

and CA forms of ROP showed different phenotypes (Molendijk et al., 2001; Jones et 537  

al., 2002; Lei et al., 2015). This discrepancy can be explained by the notion that ROP 538  

GTPases function as molecular switches. Because the homeostasis of their activity is 539  

strictly regulated, either stronger or weaker activity will affect their function. 540  

Although LjSPK1 can interact with three ROP GTPases, only one of them, LjROP6, 541  

is required for the polar elongation of ITs in root hairs. Perhaps LjROP6 interacts with 542  

LjNFR5 to transduce NF signaling to mediate the progression and elongation of ITs. 543  

Interestingly, in a study of M. truncatula ROP10, only the MtROP10 CA form 544  

showed defects in the polarized growth of root hairs; its infection phenotype was very 545  

similar to those of the LjROP6 CA and LjROP6 DN forms. However, although 546  

MtROP10 was able to interact with the NF receptor MtNFP and it was required for 547  

root hair deformation, it was not required for the polarized growth of ITs (Lei et al., 548  

2015). Therefore, the current and previous findings suggest that different ROP 549  

GTPases that are associated with NF signaling mediate different infection processes. 550  

Prenylation is required for the membrane attachment and function of type I ROP 551  

GTPases, and LjROP6 is a type I ROP GTPase. When we examined the subcellular 552  

localization of LjROP6 in N. benthamiana, the placement of the fluorescent protein at 553  

the N-terminus or C-terminus of this protein did not affect its PM localization. 554  

Moreover, in the complementation assay, LjROP6 tagged with mCherry at its 555  

C-terminus fully rescued the infection phenotype of Ljrop6-1. These results suggest 556  

that the prenylation of LjROP6 has minor effects on its function, which are consistent 557  

with the results of another study in Arabidopsis (Sorek et al., 2011). 558  

The penetration of bacteria into legume roots is a key step in the specific 559  

recognition of compatible rhizobia during the formation and progression of ITs. The 560  

current and previous findings provide genetic evidence that the molecular machinery 561  

associated with the LjNFR5-LjROP6-LjSPK1 module is involved in the vesicle 562  

trafficking and cytoskeleton rearrangements required for the polarized growth of ITs. 563  

Thus, we suggest that the LjNFR5-LjROP6-LjSPK1 module has been co-opted to 564  

participate in some events in rhizobial infection, particularly the polarized growth of 565  

ITs (Supplemental Figure 11A and B). Our results also show that LjSPK1 can activate 566  

other type I ROP GTPases such as LjROP1 and LjROP3 and that these ROP GTPases 567  

are required for the growth of the root hair tip (Supplemental Figure 11C). 568  

569  

METHODS 570  

Biological Materials and Strains 571  

Lotus japonicus accession Gifu B-129 was used in this study. The Ljrop and Ljspk1 572  

mutants were obtained from the LORE1 transposon insertion library at Lotus Base 573  

(https://lotus.au.dk) (Fukai et al., 2012;   Urbanski et al., 2012). The mutants were 574  

genotyped by PCR and sequenced using the primers shown in Supplemental Data Set 575  

1. 576  

The rhizobium strain Mesorhizobium loti R7A carrying pXLGD4 (lacZ), 577  

pMP2444 (GFP), or mTag was used for infection and the nodulation assays. The 578  

Agrobacterium rhizogenes strain AR1193 was used for hairy root transformation of L. 579  

japonicus and M. truncatula roots, and the A. tumefaciens strain EHA105 was used 580  

for transient expression in N. benthamiana. Plasmids were transformed into 581  

Escherichia coli DH10B or DH5α for cloning or into E. coli Rosetta (DE3) for protein 582  

expression. The yeast strain NMY51 was used for the DUAL membrane yeast 583  

two-hybrid system. 584  

585  

Cloning, DNA Manipulation, and Plasmid Construction 586  

The coding sequences (CDS) of the LjROPs and LjSPK1 were amplified from the 587  

Gifu cDNA library by PCR amplification; the CA and DN forms of LjROPs were 588  

generated with a Hieff MutTM Site-Directed Mutagenesis Kit (Yeasen Biotechnology, 589  

Shanghai, China) as per the manufacturer’s instructions. 590  

For expression analysis of LjSPK1 in L. japonicus hairy roots,   the LjSPK1 591  

promoter (2 kb upstream of the start codon) was amplified from Gifu genomic DNA 592  

by PCR. The PCR product was inserted into pDONR207 by BP reaction (Invitrogen), 593  

and combined into pKGWFS7 to generate the pLjSPK1:GUS construct by LR 594  

reaction (Invitrogen). 595  

For the LjSPK1 RNAi construct, the CDS fragment of LjSPK1 was amplified by 596  

PCR. The PCR product was inserted into pDONR207 by BP reaction (Invitrogen) and 597  

combined into pUB-GWS-GFP to generate the LjSPK1-Ri construct by LR reaction 598  

(Invitrogen). 599  

To overexpress LjSPK1 or LjROPs in L. japonicus hairy roots, the LjSPK1, 600  

LjROPs, LsROPs CA, and LjROPs DN CDS were transferred from X-pDONR207 into 601  

pUB-GW-GFP by LR reaction to generate the LjSPK1-OX, LjROPs-OX, LjROPs CA, 602  

or DN constructs, respectively. 603  

For the DUAL membrane yeast two-hybrid system, the PCR products and 604  

pCCW-STE or pDSL-Nx were digested with Sfi1 and the LjROPs and LjSPK1-DHR2 605  

were inserted into pCCW-STE and pDSL-Nx, respectively, using T4 DNA ligase 606  

(Takara, Dalian, China). 607  

To measure guanine nucleotide exchange activity, the LjROP6 and 608  

LjSPK1-DHR2 PCR products were recombined into pDONR207 by BP reaction 609  

(Invitrogen). LjROP6 pDONR207 and LjSPK1-DHR2 pDONR207 were recombined 610  

into pHGWA with a His-tag or pGGWA with a GST-tag by LR reaction and 611  

transformed into E. coli Rosetta for protein expression. 612  

For the luciferase biomolecular complementation assays in N. benthamiana, the 613  

PCR products and luciferase vector pCambia1300-35S-nLuc or 614  

pCambia1300-35S-cLuc were digested with Kpn1 and Sal1, and LjSPK1-DHR2 and 615  

LjROP6 were inserted into pCambia1300-X-nLuc or pCambia1300-cLuc-X using T4 616  

DNA ligase. 617  

For co-immunoprecipitation assays in N. benthamiana, the PCR products 618  

(LjROP6, LjROP6 CA, LjROP6 DN, or LjROP10) were inserted into destination 619  

vector pCambia1305-35S-Myc following Kpn1 and Sal1 digestion. The 620  

LjSPK1-DHR2 PCR products were inserted into destination vector 621  

pUB-GFP/X-FLAG, which was modified from pUB-GFP (Maekawa et al., 2008; Li 622  

et al., 2019), following Kpn1 and Asc1 digestion. 623  

To determine the subcellular localization of LjROP6 in L. japonicus leaves and L. 624  

japonicus hairy roots, LjROP6 pDONR207 was recombined into pK7WGF2 to 625  

generate GFP-LjROP6 by LR reaction. LjROP6 PCR products were inserted into 626  

pUB-GFP/X-mCherry (this vector was modified from pUB-GFP) (Li et al., 2019) 627  

following Xba1 and Kpn1 digestion using T4 DNA ligase to generate 628  

LjROP6-mCherry. 629  

For the complementation assay via hairy root transformation of Ljrop6-1, the 630  

LjROP6 promoter (1.8Kb upstream of the start codon) was amplified from Gifu 631  

genomic DNA by PCR amplification. The PCR products and destination vector 632  

pUB-GFP were digested with Pst1 and Xba1, and the LjROP6 promoter was inserted 633  

into pUB-GFP using T4 DNA ligase to generate pLjROP6-GFP. pLjROP6-GFP and 634  

pUB-GFP/LjROP6-mCherry were digested with Xba1 and Asc1, and 635  

LjROP6-mCherry was inserted into pLjROP6-GFP using T4 DNA ligase to generate 636  

pLjROP6:LjROP6-mCherry. This vector was also used to determine the subcellular 637  

localization of LjROP6 in L. japonicus transformed hairy roots. 638  

To determine the subcellular localization of LjSPK1 in N. benthamiana leaves, 639  

the LjSPK1 PCR products were inserted into pCambia1300-35S-YFP following Sma1 640  

and Spe1 digestion to generate LjSPK1-YFP. For LjSPK1 localization and BiFC 641  

analysis in N. benthamiana leaves, L. japonicus, or M. truncatula hairy roots, the 642  

LjSPK1-eGFP and BiFC constructs were generated by Golden Gate cloning (Peccoud 643  

et al., 2011). The LjSPK1 CDS was synthesized in the level 0 vector pL0V-SC3 644  

(Shanghai Xitubio Biotechnology Co., Ltd.). This vector and the EC16570 vector 645  

were digested with Bsa1 to generate LjSPK1-eGFP as the level 1 construct. This level 646  

1 LjSPK1-eGFP was assembled into EC50507 (https://www.ensa.ac.uk/), adding 647  

p35S:NLS-DsRed as a transgenic marker, to generate the level 2 construct 648  

LjSPK1-eGFP. The synthesized LjSPK1 CDS in vector pL0V-SC3 was assembled 649  

into EC12850 to generate LjSPK1-cVenus; the LjROP6 PCR product was inserted 650  

into pL0V-SC3 following Bbs1 digestion, and this construct was assembled into 651  

EC12849 to generate LjROP6-nVenus. Finally, these constructs were assembled into 652  

EC50507, adding p35S:DsRed as a transgenic marker, to generate the BiFC construct 653  

pLjUb:LjSPK1-cVerns/pAtUb:LjROP6-nVenus. 654  

All PCR amplification was carried out using high-fidelity DNA polymerase 655  

KOD Plus (Toyobo, Osaka, Japan) or MAX (Vazyme, Nanjing, China), and all 656  

constructs were confirmed by DNA sequencing. The constructs in destination vectors 657  

were introduced into A. rhizogenes AR1193 for hairy root transformation in L. 658  

japonicus or M. truncatula or into A. tumefaciens strain EHA105 for transient 659  

expression in N. benthamiana by electroporation. All primers are listed in 660  

Supplemental Data Set 1, and all constructs are listed in Supplemental Table 1. 661  

662  

Analysis of Plant Growth, Rhizobial Inoculation, Infection, Nodulation, Root 663  

Hairs, and Actin Rearrangement 664  

L. japonicus seeds were scarified, surface sterilized in 10% (v/v) sodium hypochlorite 665  

for 7 min, and rinsed five times with sterile water. The sterilized seeds were imbibed 666  

in water, transferred to 0.8% (v/v) water agar plates, and grown on 22°C in the dark 667  

for 3–4 days for germination. The seedlings were transferred to a mixture of perlite 668  

and vermiculite (1:1), cultivated in a growth chamber under a 16 h/8 h light (250 669  

µmol/m2/s)/dark cycle, and inoculated with rhizobia at 5–7 days after transfer. The 670  

infection phenotypes and events were determined at the indicated time points by laser 671  

scanning confocal microscopy (FV1000, Olympus, Tokyo, Japan) using GFP-marked 672  

M. loti R7A, or light microscopy (ECLIPSE Ni, Nikon, Tokyo, Japan) after staining 673  

the roots with 5-bromo-4-chloro-3-indolylbeta-D-galacto-pyranoside (X-Gal).  674  

To prepare nodule sections, the nodules were fixed in glutaraldehyde (2.5% v/v), 675  

embedded in Technovit 7100 resin (Kulzer GmbH, Wehrheim, Germany) according 676  

to the manufacturer’s instructions, and cut into 5–10 mm transverse sections with a 677  

microtome (RM2265, Leica Microsystems, Wetzlar, Germany). Root nodule sections 678  

were co-stained with toluidine blue or Magenta-Gal to visualize GUS or M. loti. 679  

To analyze the root hair phenotypes of the Ljrop mutants, the seedlings were 680  

transferred to glass slides containing 1 ml liquid FP medium and incubated overnight. 681  

The seedlings were inoculated by adding fresh FP medium with or without M. loti 682  

R7A (OD600 ~0.01) and incubated in the dark for ~18 h before analysis. The root 683  

hairs were observed and imaged under a light microscope (Nikon ECLIPSE Ni). For 684  

observation root hairs in L. japonicus hairy roots, transformed hairy roots of the 685  

LjROPs-overexpression, LjROPs CA, and LjROPs DN lines were scored by GFP 686  

fluorescence using a Nikon SMZ1500 microscope, and then root hairs were observed 687  

and imaged under a light microscope (Nikon ECLIPSE Ni). The length of root hairs 688  

was measured using ImageJ. Five root hair cells were measured per transformed root, 689  

and at least ten transformed roots were scored. 690  

Phalloidin staining was done as described previously (Yokota et al., 2009), and 691  

actin was observed by laser scanning confocal microscopy (FV1000, Olympus, Tokyo, 692  

Japan). 693  

All assays were validated in at least three independent experiments. 694  

695  

Analysis of Promoter:GUS, RNAi, Overexpression, and Complementation in L. 696  

japonicus Transformed Hairy Roots 697  

The indicated constructs in A. rhizogenes AR1193 were introduced into L. japonicus 698  

Gifu or Ljrop6-1 roots on 1/2 B5 medium via hairy root transformation. After 2 weeks, 699  

the transformed hairy roots were scored by GFP fluorescence, as observed under a 700  

Nikon SMZ1500 microscope. The transformed chimeric plants were transferred to 701  

pots filled with a mixture of vermiculite and perlite (1:1) and inoculated with M. loti 702  

R7A/lacZ at 5-7 days after transfer. For pLjSPK1:GUS in L. japonicus hairy roots, the 703  

hairy roots were stained with both Magenta-Gal and X-Gluc to visualize M. loti R7A 704  

and gene expression at the indicated time points. For the LjSPK1-Ri, LjSPK1-OX, and 705  

LjROP6 complementation assays, the transgenic roots were stained with X-Gal, and 706  

the infection events were counted under a Nikon ECLIPSE Ni light microscope at 5 or 707  

7 days after inoculation. At least seven plants were analyzed per experiment. The 708  

number of nodules was counted at 2 or 3 weeks after inoculation with M. loti 709  

R7A/lacZ using at least nine plants at each time point. These assays were repeated in 710  

three individual experiments. 711  

712  

RNA Extraction and RT-qPCR 713  

Total RNA was extracted from plant roots with TRIpure Reagent (Aidlab 714  

Biotechnologies, Beijing, China) according to the manufacturer’s instruction and 715  

quantified using a Nanodrop 2000 instrument (Thermo Scientific). Reverse 716  

transcription of cDNA was performed using TransScript® One-Step gDNA Removal 717  

and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). RT-PCR was 718  

conducted using SYBR® Green Real time PCR Master Mix (Toyobo, Osaka, Japan), 719  

and the products were detected using the ABI StepOnePlus PCR system 720  

(ThermoFisher). Amplification conditions consisted of an initial denaturation step at 721  

95°C for 20 s, 40 cycles at 95°C for 5 s and 60°C for 30 s, followed by a melting 722  

curve stage of 95°C for 15 s, 60°C for 60 s and 95°C for 10 s. Ubiquitin 723  

(Lj5g3v2060710.1) was used as an internal control, and RT-qPCR data for each 724  

sample were normalized to the respective Ubiquitin expression level. Standard errors 725  

and statistical significance based on three biological replicates were calculated using 726  

the 2-ΔΔCt method. The primers used are shown in Supplemental Data Set 1. 727  

728  

Scanning Electron Microscopy 729  

The leaves of the spk1 mutants were fixed in FAA overnight at 4°C and dehydrated in 730  

in an ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol) for 5 731  

minutes per step at room temperature. The leaves were dried using a carbon dioxide 732  

critical point drier (LEICA CPD300), transferred onto a copper mount for ion 733  

sputtering, and imaged under a scanning electron microscope (JSM-6360LV, JEOL, 734  

Tokyo, Japan) using 1 to 5 KV accelerating voltage. 735  

736  

Phylogenetic Analysis 737  

The A. thaliana, M. truncatula, and L. japonicus protein sequences were obtained 738  

from the genome databases at TAIR, M. truncatula Mt4.0, and L. japonicus 739  

miyakogusa.jp website Version 3.0. Soybean (G. max), common bean (P. vulgaris) 740  

and pea (Pisum sativum) SPK1 protein sequences were obtained from   Legume 741  

Information System (https://www.legumeinfo.org/). Rice (O. sativa) OsSPK1, Homo 742  

sapiens DOCK180, nematode (Caenorhabditis elegans) CED-5, fruit fly (Drosophila 743  

melanogaster) MYOBLAST CITY (MBC), yeast (Saccharomyces cerevisiae) 744  

Ylr422wp, and slime mold (Dictyostelium discoideum) DocA protein sequences were 745  

obtained from NCBI. All protein sequences were aligned using ClustalW, and the 746  

phylogenetic tree was built using the maximum likelihood method in MEGA7. The 747  

percentage of trees in which the associated taxa clustered together is shown next to 748  

the branches. Initial trees for the heuristic search were obtained automatically by 749  

applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances 750  

estimated using a JTT model and selecting the topology with superior log likelihood 751  

value.   The tree is drawn to scale, with branch lengths representing the number of 752  

substitutions per site.  Sequence alignments of SPK1 proteins and ROP GTPases are 753  

shown in Supplemental File 1 and 2, respectively. 754  

755  

DUAL Membrane Yeast Two-Hybrid Assays 756  

The yeast strain NMY51 was transformed with the constructs in destination vectors as 757  

per manufacturer’s instructions using lithium acetate transformation (Yeast Protocols 758  

Handbook PT3024-1, Clontech, Palo Alto, CA, USA). The transformants were grown 759  

on SD (0.67% yeast nitrogen base, 2% glucose, 2% Bacto-agar and amino acid mix) 760  

without the appropriate auxotrophic markers and in the presence of 761  

3-amino-1,2,4-triazole (3-AT) at different concentrations. These assays were repeated762  

three times. 763  

764  

Luciferase Biomolecular Complementation Assays 765  

cLuc-LjROP6, cLuc-LjROP6 CA, or cLuc-LjROP6 DN was co-expressed with 766  

LjSPK1-DHR2-nLuc in N. benthamiana leaves via agroinfiltration along with p19, 767  

which inhibits gene silencing (Voinnet et al., 2003). The transformed plants were 768  

grown in a growth chamber. After 2–3 days, Luc images were captured by a CCD 769  

(Tanon, Shanghai, China) after spraying the leaves with 1 mM luciferin. All images 770  

were acquired using the same exposure settings. Each interaction group was validated 771  

in at least three replicates, and three independent experiments were performed. 772  

773  

Bimolecular Fluorescence Complementation (BiFC) Assays 774  

The LjSPK1 and LjROP6 BiFC construct 775  

(pLjUb:LjSPK1-cVenus/pAtUb:LjROP6-nVenus) was expressed in N. benthamiana 776  

leaves by agroinfiltration, along with p19. The transformed plants were grown in a 777  

growth chamber, and 2-3 days later, the images were captured by laser scanning 778  

confocal microscopy (TCS SP8, Leica). Leaves were immersed in 30% sucrose for 30 779  

min for the plasmolysis experiment. The LjSPK1-LjROP6 BiFC construct was also 780  

expressed in wild-type L. japonicus or M. truncatula sunn-1 by hairy root 781  

transformation. The transgenic hairy roots were scored based on NLS-DsRed marker, 782  

and inoculated with M. loti R7A/mTag or Rm1021/mCherry (OD600 ~0.01). The 783  

images were analyzed at 5-7 days after inoculation. The filter sets for excitation (ex) 784  

and emission (em) were as follows: Venus (ex/em, 514 nm/524–545 nm), 785  

mCherry/DsRed (ex/em, 561 nm/600–630 nm) and mTag (ex/em, 405 nm/415–454 786  

nm). All BiFC experiments were repeated twice, and at least five leaves or roots were 787  

analyzed each time. 788  

789  

Subcellular Localization in L. japonicus Transformed Hairy Roots and N. 790  

benthamiana Leaves 791  

To determine the subcellular localization of LjSPK1 and LjROP6 in L. japonicus 792  

hairy roots, the indicated constructs in A. tumefaciens strain AR1193 were expressed 793  

in L. japonicus hairy roots, and the transgenic roots were inoculated by M. loti 794  

R7A/mTag. The subcellular localization of the proteins was observed by laser 795  

scanning confocal microscopy (TCS SP8, Leica). The filter sets for excitation (ex) 796  

and emission (em) were as follows: GFP (ex/em, 488 nm/505–550 nm), 797  

FM4-64/mCherry/DsRed (ex/em, 561 nm/600–630 nm) and mTag (ex/em, 405 798  

nm/415–454 nm). 799  

To observe the subcellular localization of LjSPK1 and LjROP6 in N. 800  

benthamiana leaves, constructs in A. tumefaciens strain EHA105 were expressed in N. 801  

benthamiana leaves by agroinfiltration along with p19. Agrobacterium cells were 802  

grown overnight, collected, resuspended in buffer (10 mM MgCl2, 10 mM MES, pH 803  

5.6, and 150 µM acetosyringone), adjusted to OD600 (about 0.4), and incubated at 804  

28 °C for 2 h before infiltration. Three or four leaves were selected for injection. 805  

Following injection, the plants were kept in a growth chamber under a 16 h/8 h light 806  

(250 µmol/m2/s)/dark cycle. After 2-3 days, the leaves were observed by confocal 807  

microscopy (Olympus FV1000 or Leica TCS SP8). Leaves were immersed in 30% 808  

sucrose for 30 min for plasmolysis or FM4-64 solution for co-localization with PM. 809  

The filter sets were as follows: GFP (ex/em, 488 nm/505–550 nm), YFP (ex/em, 514 810  

nm/524–545 nm), FM4-64/mRFP/mCherry (ex/em, 561 nm/600–630 nm). Gray 811  

values were analyzed using ImageJ. We used GFP beads (SMART, CAT.SA070001) 812  

for immunoprecipitation, and GFP antibody (Abmart, CAT.M20004) and mCherry 813  

antibody (AffinitY, CAT.T0090) for immunoblot analysis. These assays were 814  

validated with three independent experiments. All protein subcellular localization 815  

assays were repeated in at least three independent experiments. 816  

817  

Protein Extraction and Co-Immunoprecipitation Assays 818  

Constructs in different combinations (ROP6-Myc, ROP6 CA-Myc, ROP6 DN-Myc, 819  

ROP10-Myc, and SPK1-Flag) were transformed into N. benthamiana leaves by 820  

agroinfiltration along with p19. The plants were grown in a growth room for 48 h 821  

under a 16 h/8 h light (250 µmol/m2/s)/dark cycle. Approximately 0.3 g leaf tissue 822  

was harvested, and leaf proteins were extracted with 1 ml extraction buffer (50 mM 823  

TRIS-MES pH 8.0, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, 5 mM DTT, 1 mM 824  

PMSF, protease inhibitor cocktail CompleteMini tablets). The mixture was 825  

centrifuged at 10,000 × g at 4°C for 15 min, and the supernatant was collected for 826  

co-immunoprecipitation assays. We used Myc beads (Abmart, CAT.M20012L) for 827  

immunoprecipitation, and Flag antibody (HuiOu Biotech, CAT.HOA012FL01) and 828  

Myc antibody (Abmart, CAT.M2000M) for immunoblotting. These assays were 829  

validated with three independent experiments. 830  

831  

Guanine Nucleotide Exchange Activity Assay 832  

To investigate the guanine nucleotide exchange activity of ROP6 and determine how 833  

it is affected by SPK1, the constructs in destination vectors described above were 834  

introduced into E. coli Rosetta cells by electroporation. The guanine nucleotide 835  

exchange assay was essentially carried out as described previously, with small 836  

modifications (Gu et al., 2006; Wang et al., 2017). The guanine nucleotide exchange 837  

activity of ROP6 at different concentrations and the effect of SPK1 on this activity 838  

were determined by monitoring fluorescence using a Varioskan Flash (Thermo 839  

Scientific, Waltham, MA, USA). The changes in fluorescence intensity were recorded 840  

every 4 s for 600 s (ex/em 360 nm/440 nm). This assay was repeated three times. 841  

842  

Statistical Analyses 843  

Statistical significance was calculated by two-tailed Student’s t-test (*P < 0.05, **P < 844  

0.01) and error bars indicate SD. Vertical box plots were generated using GraphPad 845  

Prism 6.0 software. For each box plot, the line in the box represents the median; the 846  

boxes show the upper and lower quartiles; the whiskers represent the maximum and 847  

minimum values. One-way ANOVA followed by Tukey’s multiple comparisons test 848  

was used to determine the differences. Values of P < 0.05 were considered to be 849  

statistically significant. The results of statistical analyses are shown in Supplemental 850  

Data Set 2. 851  

852  

Accession Numbers 853  

Sequence data from this article can be found in the GenBank data libraries under 854  

accession numbers MT701563 for LjSPK1 and the Lotus Base Gifu v1.2 855  

(https://lotus.au.dk/) for LotjaGi3g1v0299000. The LjROP GTPase sequences can be 856  

found in miyakogusa.jp V3.0 (http://www.kazusa.or.jp/lotus/) under the following 857  

accession numbers: LjROP1 (Lj1g3v3331690), LjROP3 (Lj2g3v1670990), LjROP5 858  

(Lj3g3v3453430), LjROP6 (Lj0g3v0167719), and LjROP10 (Lj1g3v0415230). 859  

860  

Supplemental Data 861  

Supplemental Figure 1. Phylogenetic Analysis of DOCK family GEF Proteins and 862  

Growth phenotypes of Ljspk1 Mutants. 863  

Supplemental Figure 2. Symbiotic Phenotypes of Wild Type (Gifu) and Ljspk1 864  

Mutants. 865  

Supplemental Figure 3. Interaction between LjSPK1 and L. japonicus ROP GTPases 866  

in Yeast Cells, and the Transcription Levels and Infection Events of Ljrop Mutants. 867  

Supplemental Figure 4. LjROP6 Transcript Levels and Nodulation Phenotypes of 868  

Ljrop6 Mutants. 869  

Supplemental Figure 5. Root Hair Phenotypes of Ljrop6-2 and Ljrop6-3 Mutants. 870  

Supplemental Figure 6. LjROP6 Protein Subcellular Localization in N. benthamiana 871  

Epidermal Leaf Cells and Wild-type L. japonicus Hairy Roots. 872  

Supplemental Figure 7. LjSPK1 Subcellular Localization in Ljrop6-1 and BiFC 873  

Assay of LjSPK1 and LjROP6 interactions in L. japonicus Wild-type and Mtsunn-1 874  

Hairy Roots. 875  

Supplemental Figure 8. Root Hairs Growth and Rhizobial Infection in 876  

LjROP6-Overexpression, LjROP6 CA, and LjROP6 DN Lines. 877  

Supplemental Figure 9. Root Hair Phenotypes of LjROP1/3-Overexpression, CA, 878  

and DN Lines. 879  

Supplemental Figure 10. Actin Filaments Are Disordered in Ljrop6-1 Root Hairs 880  

Compared with Wild Type. 881  

Supplemental Figure 11. A Proposed Model of LjSPK1-LjROP Function in 882  

Rhizobial Infection Thread Formation and Polarized Growth of Root Hairs. 883  

Supplemental Table 1. Constructs used in this study. 884  

Supplemental Data Set 1. Primers used in this study 885  

Supplemental Data Set 2. ANOVA and Student’s t-test Results for the Data Shown 886  

in the Figures. 887  

Supplemental File 1. Sequence Alignment of SPK1 Proteins. 888  

Supplemental File 2. Sequence Alignment of ROP GTPase Proteins. 889  

890  

ACKNOWLEDGMENTS 891  

We thank Prof. Phil Pool (U. Oxford, UK) for kindly providing us with the M. loti 892  

R7A/mTag strain, Prof. Jeremy Murray (CEMPS, CAS, China) for sharing the 893  

Golden Gate vectors, and Dr. Yoji (PSB, CAS, China) and Dr. Zhao Liu (Hebei U. of 894  

Chinese Medicine) for suggestions about GEF enzyme activity. We thank Yongfei 895  

Wang (CEMPS, CAS, China) for helpful discussions on this study. This work was 896  

funded by grants from The National Key R & D Program of China 897  

(2016YFA0500500), The Strategic Priority Research Program of Chinese Academy 898  

of Sciences (XDB27040208), and The International Partnership Program of CAS 899  

(153D31KYSB20160074). 900  

901  

AUTHOR CONTRIBUTIONS 902  

J. Liu and F. Xie designed the experiments, J. Liu performed most of the experiments,903  

M.X. Liu and L. P. Qiu isolated the rop6-1 and   spk1-1 mutants, respectively. J. Liu904  

and F. Xie analyzed the data and wrote the manuscript. 905  

906  

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Figure 1. LjSPK1 Is Induced by Nod Factors and Is Specifically Expressed in Young Nodules. (A) RT-qPCR analysis of LjSPK1 transcript levels in roots of wild-type L. japonicus (Gifu) after inoculationwith purified Nod factor (0 h, 6 h, 12 h). Expression is relative to that in mock-treated samples (0 h) andnormalized to that of Lotus Ubiquitin. Mean and SD were derived from two biological replicates. Statisticalsignificance (*P < 0.05) was evaluated by Student’s t test.(B–C) pLjSPK1:GUS expression pattern in wild-type roots without rhizobial inoculation.(D–H) pLjSPK1:GUS expression pattern in wild-type roots after inoculation with M. loti R7A expressing lacZ.GUS activity was detected in nodule primordia and young nodules (D–G), and in vascular bundles ofmature nodules (H).(I–K) Section of nodules showing that pLjSPK1:GUS expression in all cell layers in young nodules (I and J),but not in mature nodules nitrogen-fixation zone (K).Transgenic roots were co-stained with X-Gluc and Magenta-Gal to visualize pLjSPK1:GUS expression (inblue) and M. loti (in purple), respectively. Bars=100 μm in (B–H) and 1 mm in (I–K).

Figure 2. Infection Threads in Ljspk1 Mutants Are Abnormal Compared with Wild-type L. japonicus. (A–D) Normal elongating infection threads in the wild type (Gifu) (A) and typical abnormal infection events in the Ljspk1 mutants (B and C) at 1 week after inoculation with M. loti R7A/GFP. Roots were counterstained with propidium iodide before observation. Green fluorescence shows normal infection foci and a normal infection thread in a curled root hair in the wild type, whereas Ljspk1 mutants show abnormal infection processes, such as sac-like or looped ITs (B and C). Bars=10 μm. (D) A cartoon diagram of the infection events. Abbreviations: foci, infection foci; IT, infection thread in an epidermis cell; rIT, infection thread extending into a cortex cell; abnormal, abnormal IT in root hairs. (E–F) Boxplots representing the number of infection events in wild type, Ljspk1 siblings, and Ljspk1 mutants. Total number of infection events per plant (E) and number of infection events per centimeter root (F) were scored at 1 week after inoculation with M. loti R7A/lacZ. Asterisks indicate a significant difference(* for P<0.05, ** for P<0.01, Student’s t-test, comparison between wild type and mutants).(G–I) Infection thread phenotypes (G) and infection events (H–I) in wild-type L. japonicus hairy rootsexpressing control plasmid (EV), LjSPK1-Ri, or LjSPK1-OX. Total number of infection events per plant (H)and number of infection events per centimeter root (I) scored 1 week after inoculation with M. loti R7A/lacZ.Abnormal infection events are enlarged in the inset (H–I). Asterisks indicate a significant difference (* forP<0.05, ** for P<0.01, Student’s t-test, comparison between EV control and experimental group). Bars=50μm.For each boxplot: the center lines in box show the median; the box limits are the upper and lower quartiles;

the whiskers represent the maximum value and minimum value.

Figure 3. Root Hair and Infection Thread Phenotypes of Ljrop Mutants. (A) Light micrographs of root hairs in wild type (Gifu) and Ljrop mutants at 2 days after germination. Thenumbers in the lower left corners shows the number of plants with the type of root hair shown in the imageout of the total number of plants observed. Bars=100 μm.(B) Light micrograph of M. loti R7A-induced root hair deformation in the infection zone of wild type and Ljropmutants at 18 h after M. loti R7A inoculation. Bars= 100 μm.A–B: Insets (bottom) are enlargements of the areas in red boxes (top).(C–F) Infection thread phenotypes of wild type (C) and Ljrop mutants (D–F) at 5 days after inoculation withM. loti R7A/LacZ. Bars=50 μm.(G) Boxplot representing the number of infection events on roots of wild type and Ljrop mutant roots at 5days after inoculation with M. loti R7A/lacZ. The center lines in box show the median; the box limits are theupper and lower quartiles; the whiskers represent the maximum value and minimum value. Asterisksindicate a significant difference (** for P<0.01, Student’s t-test, comparison between wild type and Ljropmutants).

Figure 4. Disorientated Infection Thread Phenotypes of Ljrop6 Mutants and Complementation of Infection Thread Phenotype via Hairy Root Transformation of Ljrop6-1. (A–E) Normal infection thread phenotypes of the wild type (A) and typical misdirected infection events in all three Ljrop6 mutants (B–D). Bars=50 μm. (E) A cartoon diagram of the misdirected infection threads. Misdirected indicates misdirected or looped IT in root hairs or epidermis cell. (F–G) Boxplots representing the number of infection events in wild-type L. japonicus (Gifu) and Ljrop6 mutants at 5 days after inoculation with M. loti R7A/lacZ. Misdirected infection events are enlarged in the inset. Asterisks indicate a significant difference (* for P<0.05, ** for P<0.01, Student’s t-test, comparison between wild type and Ljrop6 mutants). (H–I) Infection thread phenotype (H) and infection events (I) of Ljrop6-1 hairy roots expressing control plasmid (EV) and pLjROP6:LjROP6-mCherry. Infection threads were stained with X-Gal and scored 5 days after inoculation with M. loti R7A/lacZ. Misdirected infection events are enlarged in the inset. Asterisks indicate a significant difference (* for P<0.05, ** for P<0.01, Student’s t-test, comparison between EV and LjROP6). Bars=50 μm. For each boxplot: the center lines in box show the median; the box limits are the upper and lower quartiles; the whiskers represent the maximum value and minimum value.

Figure 5. LjSPK1 Physically Interacts with LjROP6 and Activates its GTPase Activity in Vitro. (A) Luciferase biomolecular complementation assays showing the interaction between LjSPK1-DHR2 andLjROP6, LjROP6 CA, or LjROP6 DN in N. benthamiana leaf cells. The indicated constructs were transientlyco-expressed in N. benthamiana leaves, and luciferase complementation imaging was captured 2 daysafter agroinfiltration. nLuc, N-terminal fragment of firefly luciferase; cLuc, C-terminal fragment of fireflyluciferase. Fluorescence signal intensity is indicated.(B) Co-immunoprecipitation assay showing the interaction between LjSPK1-DHR2 and LjROP6 in N.benthamiana leaves. The indicated constructs were co-expressed in N. benthamiana leaves. The Co-IPassay was performed using anti-Myc antibody, and the proteins were detected by immunoblot analysis withanti-Flag and anti-Myc antibodies. LjSPK1-DHR2 interacted more strongly with LjROP6 DN than withLjROP6 CA. No interaction was detected between LjSPK1-DHR2 and LjROP10, a type II ROP GTPase.(C) Time course of intrinsic nucleotide exchange in LjROP6. The intrinsic guanine nucleotide exchangerates of LjROP6 increased with increasing reaction time and increasing concentration of LjROP6. Theassays contained 1 μM mant-GTP and the indicated concentration of GDP-LjROP6.(D) LjSPK1-DHR2 shows concentration-dependent GEF activity toward LjROP6. The assays contained 1μM mant-GTP, 0.5 μM GDP-LjROP6, and the indicated concentrations of LjSPK1-DHR2.Results are a representative of three independent assays with similar results.

Figure 6. Subcellular Localization of LjSPK1 in N. benthamiana Leaves and Wild-Type L. japonicus Hairy Roots. (A–F) Confocal images of LjSPK1-YFP expressed in N. benthamiana leaf cells. (A–B) Subcellular localization of LjSPK1-YFP in N. benthamiana leaves (A) and after plasmolysis via 30% sucrose treatment (B). (C) LjSPK1-YFP (green) expressed in N. benthamiana leaves and stained with the PM marker FM4-64 dye (magenta), showing that SPK1 does not merge with the PM marker. (D) LjSPK1-YFP (green) and ER marker HDEL-mRFP (magenta) were co-expressed in N. benthamiana leaves. Image shows merging of LjSPK1-YFP and HDEL-mRFP fluorescence. These genes were driven by the CaMV 35S promoter. (E–F) Intensity profiles of LjSPK1 and FM4-64 or HDEL-mRFP. Plots show fluorescence intensities of LjSPK1-YFP (green) and FM4-64 or HDEL-mRFP (magenta) in regions of interest (insets in Figure 6C–D). C–D: Insets (bottom) are enlargements of the areas in white boxes (top). Bars=10 μm. (G–J) Live cell confocal images of LjSPK1-eGFP expressed in root hairs before (G and H) and after (I and J) M. loti R7A/mTag inoculation in wild-type L. japonicus hairy roots. (G) LjSPK1-eGFP (green) fluorescencewas detected in L. japonicus hairy roots in the plasma membrane and puncta in the vicinity of the nucleus.NLS-DsRed (magenta; nuclear marker) was used as a transgenic marker. (H) LjSPK1-eGFP (green) wasdetected in L. japonicus hairy roots PM and stained with the PM marker FM4-64 dye (magenta). (I–J)LjSPK1-eGFP (green) in the PM of curled root hair (I) or with elongated IT (J) after M. loti R7A/mTaginoculaiton. IT is indicated by M. loti R7A/mTag (cyan). Asterisks indicate GFP fluorescence. Expression ofLjSPK1 was driven by the L. japonicus Ubiquitin gene promoter. Insets (right) are enlargements of the areasin white boxes (left). Bars=10 μm.

Figure 7. LjROP6 Co-localizes and Interacts with LjSPK1 at the Plasma Membrane in N. benthamiana Leaves and Hairy Roots in Legumes. (A–C) LjSPK1-YFP (green) and LjROP6-mCherry (magenta) were co-expressed in N. benthamiana leaves, showing that LjROP6 promotes LjSPK1 distribution in the plasma membrane (A). Insets (bottom) are enlargements of the areas in white boxes (top). (B) Intensity profiles of LjSPK1-YFP and LjROP6-mCherry. Plots show fluorescence intensities of LjSPK1-YFP (green) and LjROP6-mCherry (magenta) in regions of interest (insets in Figure 7A). (C) Immunoblots showing protein levels of LjSPK1 and LjROP6 in N. benthamiana leaves. Total protein was extracted and analyzed by immunoblotting with anti-GFP and anti-mCherry antibodies. Rubisco was used as the loading control. Bars=10 μm. (D–E) Bimolecular fluorescence complementation (BiFC) assay of LjSPK1-cVenus and LjROP6-nVenus expressed in N. benthamiana leaves, before (D) and after (E) plasmolysis with 30% sucrose treatment. Split Venus fluorescent was detected in the plasma membrane. Bars=10 μm. (F–H) BiFC assay of LjSPK1-cVenus and LjROP6-nVenus in wild-type L. japonicus hairy roots (F) or M. truncatula sunn-1 hairy roots (G) 5 days after rhizobial inoculation. Asterisks represent the Venus fluorescence and indicate that LjROP6 interacts with LjSPK1 in the root hair plasma membrane (F) or infection foci (G). Cyan and magenta fluorescence represent M. loti R7A/mTag (cyan, F) or Sm1021/mCherry (magenta, G), respectively. Insets (right) are enlargements of the areas in white boxes (left). Bars=10 μm.

DOI 10.1105/tpc.20.00109; originally published online October 6, 2020;Plant Cell

Jing Liu, Miaoxia Liu, Liping Qiu and Fang XieLotus japonicus

SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in

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