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

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


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

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 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   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 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 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 type and Ljrop6-1 roots. As expected, the root hairs of wild-type plants predominantly 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) 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.  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, 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). 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 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 observation root hairs in L. japonicus hairy roots, transformed hairy roots of the 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)   genetic discoveries in legume nodulation and symbiotic nitrogen fixation.