Bacterial pathogens and most nitrogen-fixing rhizobia deploy type III effectors (T3Es) as potent tools to manipulate plant signaling pathways, thereby facilitating infection and colonization. However, how rhizobial T3Es regulate legume symbiosis remains poorly understood. Here, we show that NopM, a T3E from Sinorhizobium fredii NGR234, contributes to infection and nodulation in Lotus japonicus Gifu. Loss of nopM in an NGR234ΔnopT mutant reduces infection and nodulation, whereas expression of NopM under the control of the L. japonicus NIN promoter enhances these processes. NopM associates with the Nod factor (NF) receptors NFR1 and NFR5 and physically interacts with their cytosolic domains in vitro. Notably, NopM selectively mediates the ubiquitination of NFR5. Expression of NopM in hairy roots of NFR5-HA transgenic plants correlates with increased NFR5 protein abundance compared with an inactive NopM variant. Taken together, our results indicate that NopM promotes symbiosis by increasing NFR5 abundance, thereby expanding our understanding of rhizobial T3E functions and the co-evolution of legume-rhizobium symbiosis.

It is well documented that type-III effectors are required by Gram-negative pathogens to directly target different host cellular pathways to promote bacterial infection. However, in the context of legume–rhizobium symbiosis, the role of rhizobial effectors in regulating plant symbiotic pathways remains largely unexplored. Here, we show that NopT, a YopT-type cysteine protease of Sinorhizobium fredii NGR234 directly targets the plant’s symbiotic signaling pathway by associating with two Nod factor receptors (NFR1 and NFR5 of Lotus japonicus ). NopT inhibits cell death triggered by co-expression of NFR1/NFR5 in Nicotiana benthamiana . Full-length NopT physically interacts with NFR1 and NFR5. NopT proteolytically cleaves NFR5 both in vitro and in vivo, but can be inactivated by NFR1 as a result of phosphorylation. NopT plays an essential role in mediating rhizobial infection in L. japonicus . Autocleaved NopT retains the ability to cleave NFR5 but no longer interacts with NFR1. Interestingly, genomes of certain Sinorhizobium species only harbor nopT genes encoding truncated proteins without the autocleavage site. These results reveal an intricate interplay between rhizobia and legumes, in which a rhizobial effector protease targets NFR5 to suppress symbiotic signaling. NFR1 appears to counteract this process by phosphorylating the effector. This discovery highlights the role of a bacterial effector in regulating a signaling pathway in plants and opens up the perspective of developing kinase-interacting proteases to fine-tune cellular signaling processes in general.

Besides lipo-chitooligosaccharidic Nod factors (NFs), rhizobial exo-oligosaccharides (EOS), derived from exo-polysaccharides (EPS), are symbiotic signals that promote nodule formation on legume roots. However, plant genes regulated by EOS signaling remain unidentified. Here, we used purified EPS and the rhizobial glycanase ExoK to produce EOS in vitro. EOS was applied to Lotus japonicus roots to identify genome-wide gene expression changes. Mutants of Sinorhizobium sp. NGR234 and L. japonicus were used to investigate the symbiotic roles of EOS and NFs. Transcriptomic analysis showed that EOS induced the expression of many phenylpropanoid biosynthesis genes, among other transcriptome alterations. EOS signaling occurred in both L. japonicus wild-type plants and mutants with impaired NF signaling, but not in epr3 mutant plants defective in the EOS receptor. Root exudates from EOS-treated plants showed increased genistein levels and enhanced activation of nodulation genes in NGR234, leading to higher NF production. Further experiments demonstrated that both EOS and NFs upregulate the expression of genes involved in flavonoid synthesis. Inoculation tests with NGR234 and its mutants revealed that EOS, rather than EPS, plays a role in promoting infection thread development in L. japonicus. In conclusion, we identified L. japonicus genes regulated by EOS signaling and found that EOS can stimulate the production of symbiotic phenylpropanoids that upregulate NF synthesis in rhizobia.

Legume nodulation by nitrogen-fixing rhizobia displays strict host specificity, primarily determined by rhizobial nodulation factors (NFs). While the kinase domain of NF receptors in Lotus japonicus suffices for nodule organogenesis, their extracellular domains govern rhizobial infection. In Sinorhizobium meliloti, specific NF modifications (nodL-dependent acetylation and nodF-dependent N-linked C16:2 acyl chains) are critical for infecting certain Medicago truncatula ecotypes, with LYK2bis essential for nodL-mediated infection. However, how NF receptors discern NF modifications to enable rhizobial infection remains unknown. Here, we demonstrate that M. truncatula RinRK1 (MtRinRK1) interacts with NF receptors and is indispensable for root hair infection of ecotype R108 by S. meliloti nodF nodL mutant producing modified NFs. Notably, a screen of 50 M. truncatula natural variants revealed only R108 forms functional nodules with S. meliloti nodF nodL, and we pinpointed three residues in MtRinRK1 extracellular domains essential for this infection. Despite homology with L. japonicus RinRK1 (LjRinRK1), these proteins are functionally non-interchangeable, with their extracellular domains acting as host-specific determinants. Our findings establish RinRK1 as a core regulator of NF-dependent host specificity in rhizobia infection.

CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in Medicago truncatula remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of M. truncatula mutants. Gene editing was performed for the LysM receptor kinase gene MtLYK10 and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in Nicotiana benthamiana leaves by editing a co-transformed GUSPlus gene. Transformed M. truncatula leaf explants were regenerated to whole plants at high efficiency (80%). An editing efficiency (frequency of mutations at a given target site) of up to 70% was reached in the regenerated plants. Plants with MtLYK10 knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these lyk10 mutants. Finally, the lyk10 mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing Sinorhizobium meliloti bacteria. Nodule formation was considerably delayed in the three lyk10 mutant lines. Surprisingly, the size of the rare nodules in mutant plants was higher than in wild-type plants. In conclusion, the symbiotic characterization of lyk10 mutants generated with the developed CRISPR/Cas9 protocol indicated a role of MtLYK10 in nodule formation.

Bacterial pathogens and nitrogen-fixing rhizobia employ type III protein secretion system (T3SS) effectors as potent tools to manipulate plant signaling pathways, thereby facilitating infection and host colonization. However, the molecular mechanisms underlying effects of rhizobial effectors on legume infection remain largely elusive. In this study, we investigated the symbiotic role of T3SS effectors in the interaction between Sinorhizobium fredii NGR234 and Lotus japonicus . Mutants deficient in the T3SS genes Tts1 or NopA showed enhanced rhizobial infection of L. japonicus roots. Further mutant analysis showed that the NopT effector negatively affects infection, while the NopM effector in the absence of NopT promotes infection. Notably, NopM interacts with the Nod factor receptors LjNFR1 and LjNFR5. NopM ubiquitinates LjNFR5 with ten ubiquitinated lysine residues in LjNFR5 identified using mass spectrometry. Expression of NopM in L. japonicus resulted in an approximately twofold increase in LjNFR5 protein levels and enhanced rhizobial infection. Our findings indicate that NopM directly interferes with the symbiotic signaling pathway through interaction and ubiquitination of Nod factor receptors, which likely benefits rhizobial infection of L. japonicus . Our research contributes to the intricate interplay between the Nod factor signaling pathway and rhizobial T3SS effectors delivered into host cells. ### Competing Interest Statement The authors have declared no competing interest.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs). The latter are also produced by nitrogen-fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown. Here, we analyzed the expression of a β-N-acetylhexosaminidase gene of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis. We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N-acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants. In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal.

Quorum-sensing (QS) coordinates the expression of virulence factors in Pseudomonas aeruginosa, an opportunistic pathogen known for causing severe infections in immunocompromised patients. QS has a master regulator, the lasR gene, but in clinical settings, P. aeruginosa isolates have been found that are QS-active but LasR-null. In this study, we developed an experimental evolutionary approach to identify additional QS-reprogramming determinants. We began the study with a LasR-null mutant with an extra copy of mexT, a transcriptional regulator gene that is known to be able to reprogram QS in laboratory LasR-null strains. In this strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. Using this novel method, which we have named "targeted gene duplication followed by mutant screening", we identified QS-active revertants with mutations in genes other than mexT. One QS-active revertant had a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the downregulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Our results indicate that a RpoA-dependent regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

Sinorhizobium meliloti infects the host plant alfalfa to induce formation of nitrogen-fixation root nodules, which inevitably elicit reactive oxygen species (ROS) bursts and organic peroxide generation. The MarR family regulator OhrR regulates the expression of chloroperoxidase and organic hydrogen resistance protein, which scavenge organic peroxides in free-living S. meliloti cells. The single mutant of ohrR genes SMc01945 (ohrR1) and SMc00098 (ohrR2) lacked symbiotic phenotypes. In this work, we identified the novel ohrR gene SMa2020 (ohrR3) and determined that ohrR genes are important for rhizobial infection, nodulation and nitrogen fixation with alfalfa. By analysing the phenotypes of the single, double and triple deletion mutants of ohrR genes, we demonstrate that ohrR1 and ohrR3 slightly affect rhizobial growth, but ohrR2 and ohrR3 influence cellular resistance to the organic peroxide, tert-butyl hydroperoxide. Deletion of ohrR1 and ohrR3 negatively affected infection thread formation and nodulation, and consequently, plant growth. Correspondingly, the expression of the ROS detoxification genes katA and sodB as well as that of the nitrogenase gene nifH was downregulated in bacteroids of the double and triple deletion mutants, which may underlie the symbiotic defects of these mutants. These findings demonstrate that OhrR proteins play a role in the S. meliloti-alfalfa symbiosis.

Type III protein secretion systems of nitrogen-fixing rhizobia deliver effector proteins into leguminous host cells to promote or inhibit the nodule symbiosis. However, mechanisms underlying effector-triggered inhibition of nodulation remain largely unknown. Nodulation outer protein T (NopT) of Sinorhizobium sp. NGR234 is an effector protease related to the Pseudomonas effector Avirulence protein Pseudomonas phaseolicola B (AvrPphB). Here, we constructed NGR234 mutants producing different NopT variants and found that protease activity of NopT negatively affects nodulation of smooth crotalaria (Crotalaria pallida). NopT variants lacking residues required for autocleavage and subsequent lipidation showed reduced symbiotic effects and were not targeted to the plasma membrane. We further noticed that Sinorhizobium fredii strains possess a mutated nopT gene. Sinorhizobium fredii USDA257 expressing nopT of NGR234 induced considerably fewer nodules in soybean (Glycine max) cv. Nenfeng 15 but not in other cultivars. Effector perception was further examined in NopT-expressing leaves of Arabidopsis (Arabidopsis thaliana) and found to be dependent on the protein kinase Arabidopsis AvrPphB Susceptible 1 (AtPBS1) and the associated resistance protein Arabidopsis Resistance to Pseudomonas syringae 5 (AtRPS5). Experiments with Nicotiana benthamiana plants indicated that the soybean homolog GmPBS1-1 associated with AtRPS5 can perceive NopT. Further analysis showed that NopT cleaves AtPBS1 and GmPBS1-1 and thus can activate these target proteins. Insertion of a DKM motif at the cleavage site of GmPBS1-1 resulted in increased proteolysis. Nodulation tests with soybeans expressing an autoactive GmPBS1-1 variant indicated that activation of a GmPBS1-1-mediated resistance pathway impairs nodule formation in cv. Nenfeng 15. Our findings suggest that legumes face an evolutionary dilemma of either developing effector-triggered immunity against pathogenic bacteria or establishing symbiosis with suboptimally adapted rhizobia producing pathogen-like effectors.

LasR is a master regulator of quorum-sensing (QS) in Pseudomonas aeruginosa. LasR-null mutants commonly appear in lung isolates from chronically infected cystic fibrosis (CF) patients. However, numerous such CF isolates have a QS-active phenotype, but factors underlying QS-reprogramming in LasR-null mutants remain largely unknown. Mutations in the transcriptional regulator gene mexT are well known to be responsible for QS-reprogramming in a laboratory LasR-null mutant strain, however, simultaneous occurrence of lasR and mexT mutations is rare in CF isolates. To identify QS-reprogramming determinants, we developed an experimental evolution approach, for which a QS-inactive LasR null mutant with an extra copy of mexT was engineered. In such a strain, spontaneous single mexT mutations are expected to have no or little phenotypic consequences. This novel method, named “targeted gene duplication followed by mutant screening” (TGD-MS), resulted in the identification of QS-active revertants with mutations in genes other than mexT . We characterized a QS-active revertant with a point mutation in rpoA, a gene encoding the α-subunit of RNA polymerase. QS activation in this mutant was found to be associated with the down-regulated expression of mexEF-oprN efflux pump genes. Our study therefore uncovers a new functional role for RpoA in regulating QS activity. Furthermore, our results suggest that a regulatory circuit controlling the expression of the mexEF-oprN operon is critical for QS-reprogramming. In conclusion, our study reports on the identification of non-MexT proteins associated with QS-reprogramming in a laboratory strain, shedding light on possible QS activation mechanisms in clinical P. aeruginosa isolates.

The initiation of the rhizobium-legume symbiosis requires localization of the bacteria to potential 33 infection sites on host roots. In this process, rhizobial chemotaxis and motility play a relevant role.The reviews by Aroney et al. and Compton and Scharf revised some important concepts related to 35 motility and chemotaxis and the preferred attractants of several model rhizobia. Aroney et al. 36 highlighted the importance of understanding chemotaxis and motility in legume symbionts, 37 especially under the environmental conditions found in the field. This, together with the 38 identification of different compounds sensed through the chemotaxis systems in each rhizobial 39 species, might predict symbiotic performance based on specific environmental conditions and root 40 exudate composition, making the selection of elite inoculants more accurate. 41The role of (iso)flavonoids as chemoattractants in rhizobia is not clear. However, they are crucial 42 The inter-kingdom signaling between rhizobia and legumes is complex and additional players, 87 which might not be essential for nodulation but could contribute to fine-tune some aspects of the 88

Background Protocols for Agrobacterium rhizogenes -mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus . The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL , nopM , and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1 . A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti . Conclusion We have established a novel protocol for hairy root transformation of L. japonicus . The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Exopolysaccharides (EPS) play critical roles in rhizobium-plant interactions. However, the EPS biosynthesis pathway in Bradyrhizobium diazoefficiens USDA110 remains elusive. Here we used transposon (Tn) mutagenesis with the aim to identify genetic elements required for EPS biosynthesis in B. diazoefficiens USDA110. Phenotypic screening of Tn5 insertion mutants grown on agar plates led to the identification of a mutant with a transposon insertion site in the blr2358 gene. This gene is predicted to encode a phosphor-glycosyltransferase that transfers a phosphosugar onto a polyprenol phosphate substrate. The disruption of the blr2358 gene resulted in defective EPS synthesis. Accordingly, the blr2358 mutant showed a reduced capacity to induce nodules and stimulate the growth of soybean plants. Glycosyltransferase genes related to blr2358 were found to be well conserved and widely distributed among strains of the Bradyrhizobium genus. In conclusion, our study resulted in identification of a gene involved in EPS biosynthesis and highlights the importance of EPS in the symbiotic interaction between USDA110 and soybeans.

Spodoptera litura (Fabricius) is a widely distributed, highly polyphagous pest that can cause severe damage to a variety of economically important crops. Various populations have developed resistance to different classes of insecticides. In this study, we report on two indoxacarb-resistant S. litura populations, namely Ind-R (resistance ratio = 18.37-fold) derived from an indoxacarb-susceptible (Ind-S) population and a population caught from a field (resistance ratio = 46.72-fold). A synergist experiment showed that piperonyl butoxide (PBO) combined with indoxacarb produced higher synergistic effects (synergist ratio = 5.29) in the Ind-R population as compared to Ind-S (synergist ratio = 3.08). Elevated enzyme activity of cytochrome P450 monooxygenases (P450s) was observed for Ind-R (2.15-fold) and the Field-caught population (4.03-fold) as compared to Ind-S, while only minor differences were noticed in the activities of esterases and glutathione S-transferases. Furthermore, expression levels of P450 genes of S. litura were determined by quantitative reverse transcription PCR to explore differences among the three populations. The results showed that the mRNA levels of CYP6AE68, a novel P450 gene belonging to the CYP6 family, were constitutively overexpressed in Ind-R (32.79-fold) and in the Fieldcaught population (68.11-fold). CYP6AE68 expression in S. litura was further analyzed for different developmental stages and in different tissues. Finally, we report that RNA interference-mediated silencing of CYP6AE68 increased the mortality of fourth-instar larvae exposed to indoxacarb at the LC50 dose level (increase by 33.89%, 29.44% and 22.78% for Ind-S, Ind-R and the Field-caught population, respectively). In conclusion, the findings of this study indicate that expression levels of CYP6AE68 in S. litura larvae are associated with indoxacarb resistance and that CYP6AE68 may play a significant role in detoxification of indoxacarb.

Grona triflora (Desmodium triflorum), a perennial herbaceous legume, is widely distributed in southern China. G. triflora has antipyretic, antiseptic and expectorant properties and can therefore be used as a phytomedicine (Ghosal et al. 1973). In July 2020, roots of G. triflora were investigated for nodules and rhizobia collection at the Shibaluohan Mountain Forest Park of Guangzhou. Root galls induced by a root-knot nematode were observed on 90% of the G. triflora samples (in a 200 m2 plot) and the infested plants had yellow, small and withered leaves compared with the healthy ones. The galls number on a G. triflora root ranged from 43 to 92 and the population densities of second stage juveniles (J2s) ranged from 573 to 894 per 100 cm3 soil surrounding the plant. The female perineal patterns showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between the anus and tail terminus, which matched with the description of Meloidogyne arenaria (Hartman and Sasser 1985). The J2s had the following morphometric characters (n = 15): body length = 501.05 ± 23.71 µm; body width = 17.14 ± 1.23 µm; DGO = 3.13 ± 0.27 µm; stylet length = 12.97 ± 1.38 µm; tail length = 58.02 ± 4.77 µm; hyaline tail terminus = 10.08 ± 0.65 µm. DNA from four female nematodes was isolated for PCR-based diagnostic analyses. A fragment between the COII and LrRNA genes of the mitochondrial DNA was amplified with primers C2F3/1108 (Powers and Harris 1993). In addition, a 28S ribosomal DNA D2/D3 region was amplified with primers MF/MR (Hu et al. 2011). The amplicons were sequenced (GenBank No. MW315989 and MW307358). Nucleotide BLAST results indicated that both sequences show 100% identity with corresponding M. arenaria sequences of isolates from various countries such as Brazil, China, Myanmar and Vietnam (e.g., MK033428, JQ446377, KY293688 and MK026624). For further confirmation, sequence characterized amplified region (SCAR) PCR was employed using the M. arenaria specific primers Far/Rar (Zijlstra et al. 2000). The amplicon was also sequenced (GenBank No. MW315990). The Nucleotide BLAST results showed >99% identity with M. arenaria isolates from Indonesia and Argentina (KP234264, KP253748 and MK015624). Greenhouse tests were conducted to analyze the capacity of M. arenaria to induce galls on G. triflora roots. The G. triflora seeds were collected from the sampling plot and germinated on 0.8% (W/V) agar plates. Then the seedlings were planted in 14 cm deep and 15 cm diam pots filled with sterilized soil from sampling plot. Every seedling was inoculated with 2,000 J2s (n = 15) and plants without J2s were used as a control. Two months later, galls were observed for inoculated roots while no galls were formed on roots of control plants. An average of 13,300 J2s and eggs of M. arenaria (reproduction factor = 6.65) were recovered from the root. Stanton and Rizo (1988) found that G. triflora was susceptible to M. javanica in Australia, and Ogbuji (1978) reported that a population of M. incognita reproduced on roots of G. triflora in Nigeria after artificial inoculation. To our knowledge, this is the first report on G. triflora parasitized by M. arenaria in Guangdong province. M. arenaria has potential to infest local, economically important plants like citrus, pomelo, sugarcane, maize and peanut. As G. triflora is widely distributed in southern China, there is the risk of spreading M. arenaria into agricultural and horticultural systems, that will cause yield loss and economic impacts.

Background Plant receptors with lysin motifs (LsyM) recognize microbial signals such as fungal chitin and lipo-chitooligosaccharidic Nod factors of nitrogen-fixing rhizobia. It is generally assumed that ligand-induced dimerization of LysM receptors is an essential step in activation of intracellular kinase domains and downstream signaling. Consequently, genes required for plant defense and establishment of symbiosis are expressed. We recently found that three LysM receptor proteins (namely LYK1, LYK4 and LYK5) of Arabidopsis thaliana form a tripartite receptor complex to perceive chitin. However, constitutive and ligand-induced interactions of LysM receptors generally remain difficult to be characterized. Results Interactions between ectodomains of LYK1, LYK4 and LYK5 were investigated by a chimeric receptor approach using hairy roots of the legume Lotus japonicus. Synthetic receptor pairs consisting of a LYK ectodomain and the intracellular domain of a L. japonicus Nod factor receptor (NFR1 and NFR5, respectively) were tested for their capacity to activate expression of the symbiotic NIN (nodule inception) gene. The results indicated constitutive (LYK4(ED)-LYK4(ED), LYK4(ED)-LYK5(ED)) and chitin-induced interactions (LYK1(ED)-LYK1(ED), LYK1(ED)-LYK5(ED)) of the examined ectodomains. Conclusion We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.