Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system Shin Okazakia,1, Takakazu Kanekob, Shusei Satoc, and Kazuhiko Saekid,e aDepartment of International Environmental and Agricultural Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan; bFaculty of Life Sciences, Kyoto Sangyo University, Kyoto 603-8555, Japan; cKazusa DNA Research Institute, Chiba 292-0818, Japan; and dDepartment of Biological Sciences, Faculty of Science and eKyousei Science Center for Life and Nature, Nara Women’s University, Nara 630-8506, Japan Edited by Eva Kondorosi, Hungarian Academy of Sciences, Szeged, Hungary, and approved September 5, 2013 (received for review February 5, 2013) Root–nodule symbiosis between leguminous plants and nitrogen- Previous studies have shown both positive and negative effects fixing bacteria (rhizobia) involves molecular communication be- of rhizobial T3SSs on symbiosis. In the case of Mesorhizobium tween the two partners. Key components for the establishment loti, a microsymbiont of Lotus spp., deletion of its tts genes of symbiosis are rhizobium-derived lipochitooligosaccharides (Nod results in a reduction of the number of nodules formed on the factors; NFs) and their leguminous receptors (NFRs) that initiate roots of Lotus corniculatus subsp. frondosus, whereas the number nodule development and bacterial entry. Here we demonstrate of nodules on the roots of Lotus halophilus significantly increases that the soybean microsymbiont Bradyrhizobium elkanii uses the when inoculated with a T3SS-null mutant (8). Meanwhile, the type III secretion system (T3SS), which is known for its delivery of T3Es NopL and NopP of NGR234 have positive effects on virulence factors by pathogenic bacteria, to promote symbiosis. symbiosis with their host legumes (9). Both NopL and NopP are Intriguingly, wild-type B. elkanii, but not the T3SS-deficient mu- phosphorylated by plant protein kinases, and NopL can prevent tant, was able to form nitrogen-fixing nodules on soybean nfr the complete induction of pathogenesis-related defense proteins mutant En1282. Furthermore, even the NF-deficient B. elkanii mu- when expressed in Lotus japonicus roots (10). However, the tant induced nodules unless T3SS genes were mutated. Transcrip- molecular basis for T3SS-involved nodulation enhancement has tional analysis revealed that expression of the soybean nodulation- not been fully elucidated. specificgenesENOD40 and NIN was increased in the roots of En1282 Bradyrhizobium elkanii is a microsymbiont of leguminous hosts inoculated with B. elkanii but not with its T3SS mutant, suggesting such as Glycine max and Arachis hypogea, and is used as a com- that T3SS activates host nodulation signaling by bypassing NF rec- mercial inoculant for soybean in Brazil (11). We previously ognition. Root-hair curling and infection threads were not observed demonstrated that B. elkanii USDA61 nodulation on the soy- in the roots of En1282 inoculated with B. elkanii, indicating that bean cultivar Clark-rj1 was T3SS-dependent (12). Clark-rj1 is genotypically characterized as rj rj , and the rj locus is a non- T3SS is involved in crack entry or intercellular infection. These find- 1 1 1 nodulation trait that was recently reported to represent a ings suggest that B. elkanii has adopted a pathogenic system for frameshift mutation leading to a truncated NFR1 (13). Clark-rj activating host symbiosis signaling to promote its infection. 1 exhibits a nonnodulation phenotype as well as a Lotus nfr1 mu- tant (14) when inoculated with most rhizobia, with the exception symbiotic nitrogen fixation | pathogenesis | plant disease resistance | of USDA61. We therefore speculated that B. elkanii T3SS can effector compensate for NF signaling downstream of NFRs in soybean. However, it remains to be determined whether T3SS-dependent eguminous plants such as soybean and pea establish symbiosis nodulation occurs in other nfr mutant soybeans, and whether the Lwith a group of soil bacteria called rhizobia. Rhizobia induce induction of soybean symbiosis genes by B. elkanii T3SS is in- the development of root nodules in leguminous plants and fix dependent of NF signaling. The current study examined the in- atmospheric dinitrogen to ammonia, which can be effectively volvement of B. elkanii T3SS in the nodulation and infection used by the host plants. Nitrogen-fixing nodules are formed as PLANT BIOLOGY a consequence of molecular interactions between partners. For Significance example, flavonoids excreted from host plant roots interact with the rhizobial transcriptional factor NodD to induce the expression Root–nodule symbiosis between leguminous plants and rhi- of nodulation (nod) genes of rhizobia, which are required for the zobia requires rhizobial Nod factors (NFs) and their leguminous synthesis of lipochitooligosaccharides known as Nod factors (NFs) receptors (NFRs). Here we show that symbiosis in the soybean (1). The perception of NFs via cognate host receptors (NFRs) rhizobium Bradyrhizobium elkanii is promoted by the type III induces cascades of signal transduction that are required for root- secretion system (T3SS), which delivers virulence factors via hair infection and nodule organogenesis (2). pathogenic bacteria. Nodulation tests and expression analyses Besides NFs, several rhizobial traits have been reported to using mutants of both B. elkanii and soybean (Glycine max) affect symbiosis with host legumes. Among them, the type III revealed that rhizobial T3SS activates host nodulation signal- secretion system (T3SS) is known as an introducer of virulence ing in the absence of NFs and NFRs. These results suggest that factors in animal and plant pathogens (3). Pathogenic bacteria rhizobia have adopted a pathogenic system that stimulates use the system to deliver so-called type III effector (T3E) pro- their legume hosts to initiate symbiotic programs. teins directly into host cells or the external environment, where they manipulate host cellular processes to promote pathogenicity Author contributions: S.O. designed research; S.O. performed research; T.K. and S.S. con- tributed new reagents/analytic tools; S.O. analyzed data; and S.O. and K.S. wrote (4). Some rhizobia have been shown to possess T3SSs that affect the paper. the ability of rhizobia to infect host legumes (5). A distinct The authors declare no conflict of interest. characteristic of rhizobial T3SS is that its expression is induced This article is a PNAS Direct Submission. by flavonoids derived from the host legume. Flavonoids induce Data deposition: The microarray data reported in this paper have been deposited in the NodD to activate the expression of ttsI, a transcriptional acti- Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. vator of the type III secretion gene cluster (tts). TtsI sub- GSE38520). sequently activates the tts gene cluster, and proteins are secreted 1To whom correspondence should be addressed. E-mail: [email protected]. fi via T3SS (6, 7). This cascade con nes the expression of rhizobial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. T3SS during host-plant infection. 1073/pnas.1302360110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1302360110 PNAS | October 15, 2013 | vol. 110 | no. 42 | 17131–17136 Downloaded by guest on September 23, 2021 A 3 H and L). However, when both nodC and ttsI were mutated (BEttsnod), the nodulation ability of B. elkanii was mostly abol- ished (Fig. 1 A and B). These results indicate that B. elkanii is ca- pable of infecting and nodulating soybean in a T3SS-dependent manner in the absence of NFs, although the nodulation efficiency was much lower than NF-derived nodulation. Infective Properties of B. elkanii. To investigate the infective B properties of B. elkanii in the absence of NF signaling, we followed the infection process of B. elkanii strains on Enrei and En1282. In Enrei soybean, root-hair curling and infection threads were ob- served on roots inoculated with USDA61 and BErhcJ (Fig. 3 A and B), followed by the emergence of globular nodules containing infected cells (Fig. 3 E, F, I,andJ). The numbers of infection threads formed on Enrei roots were 13.6 ± 3.9 (n = 5) with USDA61 and 9.6 ± 2.4 (n = 5) with BErhcJ. By contrast, root-hair Fig. 1. Nodulation of G. max cv. Enrei (A) and En1282 (B) inoculated with curling and infection threads were absent on the roots of En1282 wild-type and mutant strains of B. elkanii USDA61 and B. japonicum soybean inoculated with both USDA61 and BEnodC (Fig. 3 C and USDA110. Nodule numbers were counted 30 d after inoculation. Nodulation D). In En1282 inoculated with USDA61, about 20% of nodules tests were performed at least three times, values shown are means obtained showed irregular shapes and contained sectored, infected regions from at least 18 plants, and error bars indicate SDs. Statistical analyses within which the infected cells were distributed unevenly in the (Student t test) were performed comparing mutants with the wild-type central nodule zone (Fig. 3 G and K). strain. *P < 0.05; **P < 0.01. Dissection of NF- and T3SS-Derived Gene Expression in Soybean by Microarray Analysis. Plant inoculation tests suggested that nodule properties of an additional nfr1 mutant derived from the soy- formation in nfr1 soybeans is dependent on B. elkanii T3SS. To bean cultivar Enrei, and compared it with the wild-type vari- investigate the molecular basis for T3SS-dependent nodulation ant. We also investigated gene expression in soybean roots and distinguish it from NFR-dependent nodulation, we exam- induced by B. elkanii T3SS, and compared this with conven- ined gene-expression profiles in soybean roots using microarray tional NF/NFR signaling. analysis. Comparative analysis of microarray data was performed Results to dissect soybean genes up- or down-regulated by NF or T3SS signaling (Fig. 4). To extract soybean genes up- or down-regu- B. elkanii nfr1 Nodulates Mutant Soybeans in a T3SS-Dependent lated by NFR signaling, we compared gene expression in the Manner. In B. elkanii, the rhcJ gene is in an operon encoding roots of Enrei and En1282 inoculated with BErhcJ (Fig.