The Mi-EFF1/Minc17998 effector interacts with the soybean GmHub6 protein to promote host plant parasitism by Meloidogyne incognita

Background: Meloidogyne incognita is the most frequently reported species from the root-knot nematode (RKN) complex responsible for causing damage in several different crops worldwide. The interaction between M. incognita and host plants involves the secretions of molecular factors from the nematode, which mainly suppress the defense response and promote plant parasitism. On the other hand, several plant elements are associated with the immune defense system that opposes nematode infection. Results: In this study, the interaction of the Mi-EFF1/Minc17998 effector with the soybean GmHub6 (Glyma.17G099100; TCP14) protein was identied and characterized in vivo and in planta. Data showed that the GmHub6 gene is upregulated by M. incognita infection in a nematode-resistant soybean cultivar (PI595099) compared to a susceptible cultivar (BRS133). Accordingly, the Arabidopsis thaliana AtHub6 mutant line (AT3G47620, orthologous gene of GmHub6 displayed normal vegetative development of the plant but was more susceptible to M. incognita. Thus, since the soybean and A. thaliana Hub6 proteins are TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP) transcription factors involved in plant development and morphogenesis modulation, owering time regulation, and the activation of the plant immune system, our data suggest that the interaction of Mi-EFF1/Minc17998 and Hub6 proteins is associated with an increase in plant susceptibility to nematode infection during parasitism. It is suggested that this interaction may prevent the nuclear localization or disturb the activity of GmHub6 as a typical transcription factor modulating the cell cycle of the plant, avoid the activation of the host’s defense response, and successfully promote parasitism. Conclusion: Our ndings indicate the potential of the Mi-EFF1/Minc17998 effector for the development of biotechnological tools based on the approaches of RNA interference and GmHub6 gene overexpression for RKN control. effector was synthesized by Epoch Life Science (Sugar Land, TX, USA), cloned into the pENTR11 vector, propagated in E. coli DH5α, and subsequently transferred to the pGADT7-AD and pGBKT7-BD destination vectors using the LR clonase system. Y2H experiments were performed using the Matchmaker TM GAL4 Two-Hybrid System 3 (Clontech) based on the GAL4 binding (BD) and transactivation (AD) domains present in these destination vectors. Both Y2H vectors were sequentially cotransformed into competent cells of the Saccharomyces cerevisiae YRG2 strain (Matα, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538) using the lithium acetate/polyethylene glycol (PEG) method. Single colonies of cotransformed yeast were grown overnight in selective yeast nitrogen base (YNB) medium in a shaking incubator at 180 rpm at 30°C. Yeast cells were diluted in fresh YNB medium to an optical dilution (OD 600 ) of approximately 1 to 0.01. Then, 100 µl of the suspension was plated on synthetic dropout medium lacking leucine, tryptophan, and histidine and containing the 3-amino-1,2,4-triazole (3-AT) His3 gene-product competitive inhibitor at 5 to 10 mM, followed by incubation at 28°C for three to ve days. The empty pGADT7-AD and pGBKT7-BD vectors were used as negative controls for protein-protein interactions, while pGADT7-AD::NIG and pGBKT7-BD::AtWWP1 were used as positive controls. The A. thaliana AtWWP1 (AT2G41020) and NIG (AT4G13350) protein interactions were previously validated by Calil et al. [92]. BiFC assays were carried out using different combinations of the A. tumefaciens GV3101 strain carrying pSITE BiFC cEFYP (GU734652) and nEYFP (GU734651) binary vectors containing the 35S:GmHub6-cYFP and 35S:Mi-EFF1/Minc17998-nYFP fusion proteins. An A. tumefaciens coculture was coinltrated into the abaxial surface of N. tabacum leaves at an OD 600 nm of 0.7 at a nal ratio of 1:1. Yellow uorescence was analyzed in epidermal cells three days after inltration using a Zeiss inverted LSM510 META laser scanning microscope equipped with an argon laser and a helium laser as excitation sources. Yellow uorescent protein (YFP) was excited at 514 nm using an argon laser, and YFP emission was detected using a 560-615-nm lter. bimolecular complementation; produced the M. incognita inoculum, performed plant inoculation, and evaluated all bioassays. MFB performed the in silico analysis and evaluated gene expression levels in nematodes and soybeans. RCT performed the data mining of 15 transcriptome libraries and the differential expression proles of the nematode effectors. MFGS, MCMS, EVSA, MELS, DF, LLPM, and FV provided intellectual input. MFB wrote the manuscript. All authors read and approved the nal version.

Soybean (Glycine max) is one of the most important agricultural commodities worldwide and is indispensable for human and animal nutrition [50,51]. However, soybean crop expansion and yields have been limited by nematode incidence [52]. The main commercial soybean cultivars are highly susceptible to nematode infections, and under ine cient nematode management, signi cant yield and economic losses are caused annually by RKNs, including M. incognita [53]. Thus, a better understanding of the molecular interactions between soybean and nematodes may allow the development of new biotechnological tools (NBTs) for RKN control [54,55]. Herein, we identi ed and validated the interaction between the Mi-EFF1/Minc17998 effector and the soybean GmHub6 protein (orthologue of AtHub6) using in vivo and in planta approaches. Curiously, our data obtained using an A. thaliana T-DNA mutant of the AtHub6 gene suggested that the disruption of AtHub6 protein function can be associated with an increase in plant susceptibility to nematode infection. Therefore, our data strongly indicate that this interaction can modulate the development of parasitized cells, prevent the activation of the immune system and, consequently, support the parasitism of the host plant.

Results
In silico analysis of the Mi-EFF1/Minc17998 effector sequence Pairwise comparisons of nucleotide and amino acid sequences showed that the Mi-EFF1/Minc17998 effector shares low identity percentage of nucleotide and amino acid sequences with other effectors that are currently better characterized, ranging from 50 to 75% (Fig. 1A) and 15 to 35% (Fig. 1B), respectively. These sequence data suggest that the Mi-EFF1/Minc17998 effector may assume a different functional role to the effectors already known during the parasitism of the host plants. Two paralogous gene for the Mi-EFF1/Minc17998 effector were identi ed in the M. incognita genome (BioProject PRJEB8714, [56]), which showed considerable homology with its corresponding Mi-EFF1/Minc17998 gene ( Figure S1), 99% and 100% (Minc3s01563g24741 paralog gene) and 92% (Minc3s06678g40162 paralog gene) sequence identity of the nucleotide and amino acid, respectively. In addition, it has been observed that the Mi-EFF1/Minc17998 effector exhibits relatively conserved orthologous genes in the other species of the Meloidogyne genus ( Figure S1), but their role as effector proteins has not been con rmed yet. Phylogenetic analysis based on nucleotide sequences showed that the Mi-EFF1/Minc17998 effector clustered with the MiPFN3 and Mj-NULG1a effectors (Fig. 1C), while amino acid sequence analysis showed that the effector grouped most closely to the Minc00469 and MiISE5 effectors (Fig. 1D). These data obtained from sequence comparisons and the analysis of phylogenetic relationships suggest that the Mi-EFF1/Minc17998 effector does not exhibit a well-de ned origin or conserved relationships with other nematode effectors. Transcriptome data mining revealed the expression pro les of the Mi-EFF1/Minc17998 effector and the Minc3s01563g24741 gene paralog in different nematode life stages. The two genes showed similar expression levels, with higher expression in the J3, J4, and female stages, while expression was lower in the egg and preparasitic J2 stages (Fig. 1E). RT-qPCR assays revealing the Mi-EFF1/Minc17998 effector expression pro le con rmed that expression was higher in the J2/J3, J3/J4, and female stages, but signi cant expression was also observed in the egg and J2 stages (Fig.   1F). These data showed that Mi-EFF1/Minc17998 gene expression is closely associated with the infection stages in the host plant.
Soybean CDS sequences were cloned into entry and destination vectors to assess the interaction with the Mi-EFF1/Minc17998 effector by in vivo and in planta protein-protein interaction assays. Yeast two-hybrid (Y2H) assays were performed with the soybean proteins and Mi-EFF1/Minc17998, and speci c protein-protein interactions were observed only with the GmHub6 protein ( Fig. 2A). The Mi-EFF1/Minc17998 effector showed speci c interaction with the GmHub6 protein in both Y2H ( Fig. 2B and C) and in planta by bimolecular uorescence complementation (BiFC) assays in tobacco (Nicotiana tabacum) (Fig. 2D). In addition, both Mi-EFF1/Minc17998 and the GmHub6 protein showed an dimerization ability, but not autoactivation (Fig. 2B). The Mi-EFF1/Minc17998 and GmHub6 interaction was considered relatively strong based on the results of the addition of the 3AT competitive inhibitor to selective medium (Fig. 2C).
In silico characterization of the soybean GmHub proteins All eight GmHub proteins studied here showed transcript accumulation in almost all plant tissues tested ( Figure S2A to S2B). In addition, their proteinprotein interaction networks were distinct, except for GmHub10 and GmHub12, which simultaneously interacted with Glyma.07G190600 (anaphasepromoting complex 4) ( Figure S2C). GmHub6 and its homologous gene (Glyma.05G027400) showed higher amino acid identity with AtHub6 (approx. 55%) and SlTCP14 (approx. 70%), while lower sequence identity (approx. 25%) was observed with other soybean GmHub proteins except for GmHub17 ( Fig. 3A). In addition, phylogenetic analysis using amino acid sequences showed that GmHub6 and its homologous gene were grouped close to the TCP transcription factors AtHub6, GmHub17, and SlTCP14 (Fig. 3B). The biological functions of the GmHub6 protein are involved in plant development and the regulation of the defense response, and the protein contains a typical TCP domain (pfam03634) and nuclear localization signal (Tables 1 and 2; Additional le 1). The protein-protein interaction network retrieved from the STRING database highlighted that GmHub6 is the core protein that interacts with numerous other proteins ( Figure S3A) similar to the AtHub6 network ( Figure S3B). These proteins from the GmHub6 network include several other TCP proteins (Table 2), but considering the orthology with AtHub6, this network of interactions maybe even larger, including dozens of proteins with highly distinct functions [38]. Curiously, GmHub6 transcripts accumulated in almost all soybean tissues and all different conditions examined, with very low accumulation being observed in the nodules under symbiotic conditions, roots under ammonia treatment, youngest roots, and seeds, in contrast to the relatively high abundance observed in leaves ( Figure S3C). In addition, the GmHub6 gene showed a positive correlation at the expression level with the Glyma.01G014900, Glyma.16G004300, and Glyma.18G296100 genes from its network in the same soybean tissues or conditions ( Figure S3D).
GmHub6 expression pro le in soybean roots during M. incognita infection RT-qPCR assays showed that the GmHub6 gene was upregulated in the axillary roots during nematode infection (at 3 dpi) only in the nematode-resistant soybean cultivar PI595099 (Fig. 3C). However, the GmHub6 expression level was similar in the noninoculated roots of both the resistant and susceptible soybean cultivars. In contrast, a higher expression level of the GmHub6 gene was observed at 25 dpi in the resistant cultivar in both mock-treated and inoculated roots. With respect to the GmHub6 expression level in the four developmental stages (stage I, II, III, and IV) of the soybean plants, signi cant differences were observed from stage I to stage IV in both soybean cultivars. Thus, in both mock-inoculated and infected plants, the GmHub6 gene expression level was nely modulated throughout plant development, which was more pronounced in the resistant soybean cultivar, mainly as a consequence of nematode infection.
M. incognita susceptibility assessment of the A. thaliana AtHub6 mutant A. thaliana AtHub6 mutant plants showed normal development, similar to that of the WT plants (data not shown). To assess whether the interaction of the Mi-EFF1/Minc17998 effector with the soybean GmHub6 protein may be associated with an increase in plant susceptibility, the AtHub6 mutant was inoculated with 250 M. incognita J2 individuals, and the evolution of parasitism was evaluated over time. The nematode penetration e ciency, post penetration development, and formation and morphology of the galls in AtHub6 mutant plants were similar to those in the WT and AtEds1 control plants. However, at 40 dpi, the AtHub6 plants showed a greater number of eggs and J2 individuals per gram of roots, a similar number of galls per gram of roots, and a higher nematode reproduction factor (NRF) compared with the WT mutant plants ( Fig. 3D to 3G). These data indicate that plants in which the AtHub6 gene was mutated were more susceptible to the nematode.

Discussion
Plants exhibit numerous mechanisms associated with defense against pathogens that are regulated in the presence or absence of pathogens to prioritize the development of the plant or the defense response [57][58][59]. The growth defense trade-off is essential to ensure plant survival and reproduction [60]. The development and defense pathways are closely related so that any disturbance in the cell cycle can trigger the plant immune system [61,62]. Initially, the root damage caused by RKN infection releases plant-derived compounds that act as damage-associated molecular patterns (DAMPs) and subsequently activate a PTI-like basal defense response [63]. Another step in PTI against RKNs may involve the recognition of PAMPs or nematode-associated molecular patterns (NAMPs), including ascarosides, cuticle, or chitin fragments [64].
In addition to inactivating host defenses, RKNs also need to modulate the cell cycle of the host plant for the successful establishment of a feeding site [8,65,66]. The RKNs are sedentary endoparasitic pathogens that spend most of their life cycle inside the roots and giant cells from J2 entry to oviposition by adult females. This infective phase usually lasts approximately 20 to 35 days for M. incognita, and effector proteins are essential to nematode infection [67,68]. Since the rst M. incognita genome sequence was reported [2,56], several effector proteins have been identi ed, and some have been characterized, but their role after their secretion in the host plant cell is still poorly understood [15,20,34,69].
In this study, we have contributed to the knowledge of the functional characteristics of the Mi-EFF1/Minc17998 effector and proposed a role of this effector in the parasitism of the host plant. Jaouannet et al. [37] and Quentin et al. [70] demonstrated that this effector is produced in the esophageal glands of parasitic juveniles, secreted within the feeding site and targeted to the nucleus, suggesting its involvement in the modulation of host cell metabolism. Herein, we showed that this effector presents low sequence identity and distant phylogenetic relationships with other well-known effectors, suggesting a speci c mode of action after delivery in the host plant. In addition, our data showed that the Mi-EFF1/Minc17998 gene is strongly upregulated during parasitism in the J2/J3, J3/J4, and female stages but is also expressed in eggs and preparasitic J2 individuals, suggesting the role of its product as a putative avirulence protein and its involvement in the formation of giant cells. A speci c protein-protein interaction between Mi-EFF1/Minc17998 and the soybean GmHub6 protein was demonstrated, and the functional disruption of the GmHub6 protein has been speculated to occur in the context of plant parasitism. Considering that the GmHub6 protein could play an essential role similar to that of AtHub6/TCP14 in the regulation of the cell cycle, plant growth and development [41][42][43][44][45][46] and the regulation of the plant's defense responses [38,42,[47][48][49], this speculation is quite plausible. Accordingly, several molecular interactions between nematode effectors and host plant proteins have already been characterized and associated with cell cycle modulation [8,65] and host defense suppression [25,30,69,[71][72][73]. In our study, the data on the EFF1/Minc17998 effector and GmHub6 protein interaction, together with the increased susceptibility of the AtHub6 mutant plants to M. incognita infection, suggest that this effector may be associated with cell cycle modulation and/or the suppression of plant defense responses. Similarly, Kim et al. [49], Li et al. [47] and Spears et al. [43] demonstrated that the A. thaliana attcp8, attcp14, and attcp15 triple mutant exhibited impaired immune responses, while Yang et al. [48] showed that the AtTCP14 protein was targeted for degradation after interaction with the P. syringae HopBB1 effector.
Stam et al. [74] showed that the Phytophthora capsici CRN12_997 effector interacts with the tomato SlTCP14 (putative ortholog of the GmHub6 and AtHub6 genes) protein, reducing the SlTCP14 association with nuclear chromatin and altering its subnuclear localization. In addition, SlTCP14 overexpression enhances plant immunity to P. capsici, while the coexpression of the CRN12_997 effector abolishes this phenotype [74]. Accordingly, our data showed that the GmHub6 gene was upregulated in response to M. incognita infection but only in the resistant soybean cultivar, suggesting that its accumulation may be mainly associated with resistance improvement in the plants. Thus, we believe that the EFF1/Minc17998 effector acts by interacting with the GmHub6 protein to primarily alter the cell cycle, which in turn activates the immune system. Subsequently, the functional disturbance of the GmHub6 protein in plant cells targeted by the nematode strongly impairs the host defense responses and allows M. incognita to complete its life cycle.
Given this hypothesis, the use of RNAi technology to target the Mi-EFF1/Minc17998 effector may be an interesting strategy for improving resistance to M. incognita in transgenic plants. This hypothesis is supported by the low genetic variability (approx. 0.02% of nucleotides) observed in protein-coding regions among different M. incognita races or isolates [75]. In addition, only slight variations in gene copy number and expression levels have been observed among different M. incognita isolates and races [76]. In contrast, the expression modulation of the GmHub6 gene (or its orthologous genes in other crops of interest) via its overexpression or targeted transcriptional modulation using the CRISPR/dCas system [77] can be evaluated (or combined with an RNAi strategy) to improve plant resistance to RKNs.

Conclusion
Several features of the M. incognita Mi-EFF1/Minc17998 effector and soybean GmHub proteins (especially the GmHub6 protein) have been highlighted, and we suggest their great importance for successful plant parasitism or plant resistance, respectively. The interaction between the Mi-EFF1/Minc17998 effector and the soybean GmHub6 protein is suggested to be a mechanism associated with a reduction in plant resistance to nematode infection via the disruption of GmHub6 activity. The high conservation of this effector in other Meloidogyne species suggests that NBTs based on RNAi could be developed to target and downregulate this effector gene in different RKN species or races. Therefore, our ndings showed that the Mi-EFF1/Minc17998 effector and the soybean GmHub6 protein are powerful targets for the development of NBTs for nematode control in crops.

Methods
In silico analysis of the M. incognita Mi-EFF1/Minc17998 effector and soybean GmHub proteins All sequences of M. incognita effector genes were retrieved from BioProject ID PRJEB8714 (sample ERS1696677) [56] from the online WormBase Parasite Database version WBPS13 [78]. Pairwise identity matrices for nucleotide and amino acid sequences were generated using Sequence Demarcation Tool Version 1.2 software [79]. Phylogenetic analyses of the M. incognita effector sequences were performed using the Phylogeny.fr web service [80]. For these analyses, sequences were aligned with MUSCLE software [81], and the alignment was curated by the Gblocks model. Then, phylogenetic analyses were performed using the maximum likelihood method with PhyML software using approximate likelihood-ratio test (aLRT) SH-like branch support and the GTR and WAG substitution models for nucleotide and amino acid sequences, respectively. Phylogenetic trees were generated and visualized with TreeDyn software, which was implemented at the same web service. Comparative genomic trees were generated from BioProject PRJEB8714 [56]  On the other hand, the sequences and characteristics of soybean genes were retrieved from G. max Wm82.a2.v1 (BioProject: PRJNA19861) [84] via the Phytozome v.12 database [85]. Conserved domains in the gene sequences were identi ed using the CDD Database from NCBI [86], annotation was con rmed by the HMMER prediction server [87], and nuclear signal localization (NLS) motifs were predicted using the NLStradamus online tool [88]. The pairwise identity matrices were generated, and phylogenetic analyses were performed as described above. The interactome network of soybean and A. thaliana hub proteins with their interacting proteins was retrieved from the STRING database v.11 platform [89]. The organ-and tissue-speci c expression of the eight GmHub genes, including the top 10 soybean proteins with which GmHub6 interacted, is presented in the heat map plot generated by the PhytoMine tool (https://phytozome.jgi.doe.gov/phytomine/begin.do) using all gene expression data in the database related to tissue-and organ-speci c expression.
Mi-EFF1/Minc17998 expression pro le determined using RT-qPCR assays  (Table S1) and GoTaq ® qPCR Master Mix (Promega, Madson, Wisconsin, USA). The qPCR conditions included an initial step at 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by a nal melting curve analysis. The relative expression of the Mi-EFF1/Minc17998 gene was normalized using Mi18S (GenBank accession U81578) [90] as an endogenous reference gene. Three biological replicates composed of one plant each were performed, and the cDNA samples were used in technical triplicate reactions. Primer e ciency and target-speci c ampli cation were con rmed on the basis of a single distinct peak in the melting curve analysis. The relative expression level (fold change) was calculated using the 2 -∆Ct or method [91].

In vivo and in planta transactivation assays for the evaluation of protein-protein interactions
Protein-protein interaction tests were performed to evaluate the interaction of the Mi-EFF1/Minc17998 effector with eight soybean hub proteins: GmHub4 (COP9 signalosome complex subunit 5), GmHub6 (TCP family transcription factor), GmHub10 (kinesin light chain), GmHub12 (APC8/anaphasepromoting complex subunit), GmHub17 (TCP family transcription factor), GmHub42 (transcription factor UNE12-related), GmHub47 (jasmonate ZIM domain-containing protein), and GmHub61 (uncharacterized conserved protein containing an emsy amine-terminus domain) ( Table 1). The cDNA sequences of the soybean hub proteins were ampli ed from total RNA isolated from the roots of the soybean cv. Williams 82. Amplicons of the expected size were cloned into the pGEMT easy vector (Promega, Madson, Wisconsin, USA) and sequenced by Macrogen (Geumcheon-gu, Seoul, South Korea); after sequence analysis, desirable amplicons were subcloned based on the restriction sites present in the primers into the entry vector of the Gateway cloning system (pENTR11; Invitrogen). Sequence identities were con rmed by comparison with gene sequences retrieved from G. max Wm82.a2.v1 (BioProject: PRJNA19861) [84] via the Phytozome v.12 database [85]. The transfer of the cDNA clones from the entry vector to the pGADT7-AD, pGBKT7-BD, and BiFC destination vectors was performed using the enzyme Gateway ™ LR Clonase ™ II Enzyme mix (Invitrogen). The full-length cDNA sequence of the Mi-EFF1/Minc17998 effector was synthesized by Epoch Life Science (Sugar Land, TX, USA), cloned into the pENTR11 vector, propagated in E. coli DH5α, and subsequently transferred to the pGADT7-AD and pGBKT7-BD destination vectors using the LR clonase system. Y2H experiments were performed using the Matchmaker TM GAL4 Two-Hybrid System 3 (Clontech) based on the GAL4 binding (BD) and transactivation (AD) domains present in these destination vectors. Both Y2H vectors were sequentially cotransformed into competent cells of the Saccharomyces cerevisiae YRG2 strain (Matα, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538) using the lithium acetate/polyethylene glycol (PEG) method. Single colonies of cotransformed yeast were grown overnight in selective yeast nitrogen base (YNB) medium in a shaking incubator at 180 rpm at 30°C. Yeast cells were diluted in fresh YNB medium to an optical dilution (OD 600 ) of approximately 1 to 0.01. Then, 100 µl of the suspension was plated on synthetic dropout medium lacking leucine, tryptophan, and histidine and containing the 3-amino-1,2,4-triazole (3-AT) His3 gene-product competitive inhibitor at 5 to 10 mM, followed by incubation at 28°C for three to ve days. The empty pGADT7-AD and pGBKT7-BD vectors were used as negative controls for proteinprotein interactions, while pGADT7-AD::NIG and pGBKT7-BD::AtWWP1 were used as positive controls. The A. thaliana AtWWP1 (AT2G41020) and NIG (AT4G13350) protein interactions were previously validated by Calil et al. [92].
BiFC assays were carried out using different combinations of the A. tumefaciens GV3101 strain carrying pSITE BiFC cEFYP (GU734652) and nEYFP (GU734651) binary vectors containing the 35S:GmHub6-cYFP and 35S:Mi-EFF1/Minc17998-nYFP fusion proteins. An A. tumefaciens coculture was coin ltrated into the abaxial surface of N. tabacum leaves at an OD 600 nm of 0.7 at a nal ratio of 1:1. Yellow uorescence was analyzed in epidermal cells three days after in ltration using a Zeiss inverted LSM510 META laser scanning microscope equipped with an argon laser and a helium laser as excitation sources. Yellow uorescent protein (YFP) was excited at 514 nm using an argon laser, and YFP emission was detected using a 560-615-nm lter.
GmHub6 expression pro le in soybean roots during M. incognita infection M. incognita J2 race 1 was obtained from tomato plants (Solanum lycopersicum cv. Santa Clara) that were inoculated and maintained for eight to ten weeks under greenhouse conditions. Infected roots were washed and macerated using a blender after treatment with 0.5% sodium hypochlorite. Eggs were harvested, rinsed with tap water, and subsequently separated from root debris using 100-to 550-μm sieves [93]. Then, the eggs were hatched under aerobic conditions at 28°C, and J2 individuals were harvested every two days, decanted and quanti ed under a microscope using a counting chamber.
Soybean plants were inoculated with 1,000 newly hatched M. incognita J2 individuals suspended in distilled water. The conventional soybean cultivars PI595099 (resistant) and BRS133 (susceptible), which are considered to exhibit contrasting RKN resistance levels [94], were inoculated with 1,000 M. incognita J2 individuals, and axillary root samples were harvested at 3, 8, 15, and 25 dpi from mock-and nematode-inoculated plants. Total RNA was puri ed using the Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA) supplemented with PVP-40, and cDNA was synthesized from DNA-free, highly pure RNA as described above. The expression pro le of the GmHub6 gene during nematode infection was measured by RT-qPCR assays using speci c primers and normalized with GmCYP18 (Glyma.12G024700) as an endogenous reference gene (Table S1). The thermocycling reactions and conditions used were the same as those described above. Four biological replicates were performed for each treatment, and each biological replicate was composed of four plants. All cDNA samples were used in technical triplicates, and primer e ciency and target-speci c ampli cation were con rmed by a single, distinct peak in the melting curve analysis. The relative expression level (fold-change) was calculated using the 2 -∆Ct method [91].
M.incognita resistance assessment of the AtHub6 mutant A. thaliana seeds from the AtHub6 gene mutant line hub6 (T-DNA insertion; attcp14-5, GK-611C04/CS458588, of AT3G47620, an orthologous gene of soybean GmHub6; Additional le 1) and the null mutant line for the enhanced disease susceptibility 1 (eds1; AT3G48090; SALK_034340) gene were obtained from the Arabidopsis Biological Resource Center (ABRC; Columbus OH, 43210, USA). The A. thaliana seeds were surfaced sterilized and sown in Murashige and Skoog (MS)-containing agar plates. The plates were strati ed in the dark at 4°C for 72 h. Plants were grown in a growth chamber at 22°C under a 12 h light/12 h dark photoperiod. For growth under in vivo conditions, plants from the WT, AtEds1, and AtHub6 lines were transferred to 1:1 substrate: sand (autoclaved commercial substrate and sand at a 1:1 ratio) and grown as described above. Then, two-or three-week-old plants were inoculated with 250 M. incognita J2 individuals as described above. The inoculated roots were harvested at 5, 10, 15, and 25 days postinoculation (dpi) and stained with acid fuchsin as described by Bybd et al. [95], and the penetration e ciency in the roots, the post penetration development of the nematodes, and the formation of galls were evaluated. In addition, the number of eggs per gram of roots, the number of J2 individuals per gram of roots, the number of galls per gram of roots, and NRF were determined from an additional plant set at 40 dpi. The NRF was determined as described above, and