CRISPR/Cas9-Mediated Generation of Mutant Lines in Medicago truncatula Indicates a Symbiotic Role of MtLYK10 during Nodule Formation.

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.

New Evolutionary Insights into RpoA: A Novel Quorum Sensing Reprograming Factor in Pseudomonas aeruginosa.

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.

An extracellular ß-N-acetylhexosaminidase of Medicago truncatula hydrolyzes chitooligosaccharides and is involved in arbuscular mycorrhizal symbiosis but not required for nodulation.

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 ß-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.

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

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.

The Medicago truncatula hydrolase MtCHIT5b degrades Nod factors of Sinorhizobium meliloti and cooperates with MtNFH1 to regulate the nodule symbiosis.

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.

Effector-triggered inhibition of nodulation: A rhizobial effector protease targets soybean kinase GmPBS1-1.

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.

Identification of an Exopolysaccharide Biosynthesis Gene in Bradyrhizobium diazoefficiens USDA110.

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.

Functional analysis of CYP6AE68, a cytochrome P450 gene associated with indoxacarb resistance in Spodoptera litura (Lepidoptera: Noctuidae).

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 Field-caught 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.

Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus.

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.

First report of Meloidogyne arenaria on roots of Grona triflora in Guangdong Province, China.

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.

Bradyrhizobium sp. strain ORS278 promotes rice growth and its quorum sensing system is required for optimal root colonization.

Many Gram-negative bacteria communicate by using homoserine lactones (HSLs) as quorum sensing (QS) signals in a cell density-dependent manner. In addition to fatty acyl-HSL (acyl-HSL) signals, certain strains, most of them associated with plants, produce non-canonical aryl-HSLs such as cinnamoyl-HSL. However, the role of aryl-HSL in endophytic associations remained elusive. Bradyrhizobium sp. strain ORS278 possesses a LuxI-LuxR type QS system and produces cinnamoyl-HSL as a QS signal. Here, we report that strain ORS278 promotes growth of domesticated rice (Oryza sativa). QS mutants unable to produce cinnamoyl-HSL exhibited reduced plant-growth promoting activity in comparison to the parent strain ORS278. Likewise, the QS mutants were impaired in their ability to colonize rice roots. These findings suggest that genes controlled by cinnamoyl-HSL play an important role in the association between rice and ORS278. However, biofilm production was not visibly altered in these mutants. In conclusion, our study highlights the importance of aryl-HSLs in endophytic plant-bacteria interactions.

NopD of Bradyrhizobium sp. XS1150 Possesses SUMO Protease Activity.

Effectors secreted by the type III protein secretion system (T3SS) of rhizobia are host-specific determinants of the nodule symbiosis. Here, we have characterized NopD, a putative type III effector of Bradyrhizobium sp. XS1150. NopD was found to possess a functional N-terminal secretion signal sequence that could replace that of the NopL effector secreted by Sinorhizobium sp. NGR234. Recombinant NopD and the C-terminal domain of NopD alone can process small ubiquitin-related modifier (SUMO) proteins and cleave SUMO-conjugated proteins. Activity was abolished in a NopD variant with a cysteine-to-alanine substitution in the catalytic core (NopD-C972A). NopD recognizes specific plant SUMO proteins (AtSUMO1 and AtSUMO2 of Arabidopsis thaliana; GmSUMO of Glycine max; PvSUMO of Phaseolus vulgaris). Subcellular localization analysis with A. thaliana protoplasts showed that NopD accumulates in nuclear bodies. NopD, but not NopD-C972A, induces cell death when expressed in Nicotiana tabacum. Likewise, inoculation tests with constructed mutant strains of XS1150 indicated that nodulation of Tephrosia vogelii is negatively affected by the protease activity of NopD. In conclusion, our findings show that NopD is a symbiosis-related protein that can process specific SUMO proteins and desumoylate SUMO-conjugated proteins.

A method for functional testing constitutive and ligand-induced interactions of lysin motif receptor proteins.

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 (LYK4ED-LYK4ED, LYK4ED-LYK5ED) and chitin-induced interactions (LYK1ED-LYK1ED, LYK1ED-LYK5ED) of the examined ectodomains. CONCLUSION: We present a method to functionally analyze constitutive and ligand-induced interactions of LysM-type proteins.

The genome of Ensifer alkalisoli YIC4027 provides insights for host specificity and environmental adaptations.

BACKGROUND: Ensifer alkalisoli YIC4027, a recently characterized nitrogen-fixing bacterium of the genus Ensifer, has been isolated from root nodules of the host plant Sesbania cannabina. This plant is widely used as green manure and for soil remediation. E. alkalisoli YIC4027 can grow in saline-alkaline soils and is a narrow-host-range strain that establishes a symbiotic relationship with S. cannabina. The complete genome of this strain was sequenced to better understand the genetic basis of host specificity and adaptation to saline-alkaline soils. RESULTS: E. alkalisoli YIC4027 was found to possess a 6.1-Mb genome consisting of three circular replicons: one chromosome (3.7 Mb), a chromid (1.9 Mb) and a plasmid (0.46 Mb). Genome comparisons showed that strain YIC4027 is phylogenetically related to broad-host-range Ensifer fredii strains. Synteny analysis revealed a strong collinearity between chromosomes of E. alkalisoli YIC4027 and those of the E. fredii NGR234 (3.9 Mb), HH103 (4.3 Mb) and USDA257 (6.48 Mb) strains. Notable differences were found for genes required for biosynthesis of nodulation factors and protein secretion systems, suggesting a role of these genes in host-specific nodulation. In addition, the genome analysis led to the identification of YIC4027 genes that are presumably related to adaptation to saline-alkaline soils, rhizosphere colonization and nodulation competitiveness. Analysis of chemotaxis cluster genes and nodulation tests with constructed che gene mutants indicated a role of chemotaxis and flagella-mediated motility in the symbiotic association between YIC4027 and S. cannabina. CONCLUSIONS: This study provides a basis for a better understanding of host specific nodulation and of adaptation to a saline-alkaline rhizosphere. This information offers the perspective to prepare optimal E. alkalisoli inocula for agriculture use and soil remediation.

Tobacco Cutworm (Spodoptera Litura) Larvae Silenced in the NADPH-Cytochrome P450 Reductase Gene Show Increased Susceptibility to Phoxim.

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductases (CPRs) function as redox partners of cytochrome P450 monooxygenases (P450s). CPRs and P450s in insects have been found to participate in insecticide resistance. However, the CPR of the moth Spodoptera litura has not been well characterized yet. Based on previously obtained transcriptome information, a full-length CPR cDNA of S. litura (SlCPR) was PCR-cloned. The deduced amino acid sequence contains domains and residues predicted to be essential for CPR function. Phylogenetic analysis with insect CPR amino acid sequences showed that SlCPR is closely related to CPRs of Lepidoptera. Quantitative reverse transcriptase PCR (RT-qPCR) was used to determine expression levels of SlCPR in different developmental stages and tissues of S. litura. SlCPR expression was strongest at the sixth-instar larvae stage and fifth-instar larvae showed highest expression in the midgut. Expression of SlCPR in the midgut and fat body was strongly upregulated when fifth-instar larvae were exposed to phoxim at LC15 (4 µg/mL) and LC50 (20 µg/mL) doses. RNA interference (RNAi) mediated silencing of SlCPR increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose). Our results provide key information on the SlCPR gene and indicate that SlCPR expression levels in S. litura larvae influence their susceptibility to phoxim and possibly other insecticides.

LYK4 is a component of a tripartite chitin receptor complex in Arabidopsis thaliana.

LysM receptor-like kinases (LYKs) of Arabidopsis thaliana (namely LYK1, LYK4 and LYK5) play a major role in chitin perception and immunity against pathogenic fungi. Chitin-induced heterodimerization of LYK1 and LYK5 has been previously reported, but protein interaction partners of LYK4 have not yet been identified. In this study, by analysing mutants we confirmed a role of LYK4 in chitin perception, and found that the ectodomain of LYK4 homodimerizes and also interacts with the ectodomain of LYK5 in vitro. Pull-down experiments with proteins expressed in protoplasts indicated LYK4-LYK4 and LY4-LYK5 interactions in planta. When protoplasts were treated with chitoheptaose or chitin, a protein complex was immunoprecipitated that appeared to be composed of LYK1, LYK4, and LYK5. Similar experiments with proteins expressed in lyk mutant plants suggested that elicitor treatment induced a physical interaction between LYK1 and LYK5 but not between LYK1 and LYK4. Bimolecular fluorescence complementation experiments substantiated these findings. Overall, our data suggest that LYK4 functions as a LYK5-associated co-receptor or scaffold protein that enhances chitin-induced signaling in Arabidopsis.

The LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE 1 protein of banana is required for perception of pathogenic and symbiotic signals.

How plants can distinguish pathogenic and symbiotic fungi remains largely unknown. Here, we characterized the role of MaLYK1, a lysin motif receptor kinase of banana. Live cell imaging techniques were used in localization studies. RNA interference (RNAi)-silenced transgenic banana plants were generated to analyze the biological role of MaLYK1. The MaLYK1 ectodomain, chitin beads, chitooligosaccharides (COs) and mycorrhizal lipochitooligosaccharides (Myc-LCOs) were used in pulldown assays. Ligand-induced MaLYK1 complex formation was tested in immunoprecipitation experiments. Chimeric receptors were expressed in Lotus japonicus to characterize the function of the MaLYK1 kinase domain. MaLYK1 was localized to the plasma membrane. MaLYK1 expression was induced by Foc4 (Fusarium oxysporum f. sp. cubense race 4) and diverse microbe-associated molecular patterns. MaLYK1-silenced banana lines showed reduced chitin-triggered defense responses, increased Foc4-induced disease symptoms and reduced mycorrhization. The MaLYK1 ectodomain was pulled down by chitin beads and LCOs or COs impaired this process. Ligand treatments induced MaLYK1 complex formation in planta. The kinase domain of MaLYK1 could functionally replace that of the chitin elicitor receptor kinase 1 (AtCERK1) in Arabidopsis thaliana and of a rhizobial LCO (Nod factor) receptor (LjNFR1) in L. japonicus. MaLYK1 represents a central molecular switch that controls defense- and symbiosis-related signaling.

Exposure to herbicides reduces larval sensitivity to insecticides in Spodoptera litura (Lepidoptera: Noctuidae).

Herbicides and insecticides are widely used in modern agriculture. It has been reported in various studies that application of insecticides can increase tolerance of herbivorous insects to insecticides. However, limited information exists on susceptibility to insecticides when insects are exposed to herbicides. This study was conducted to investigate the potential impact of the herbicides trifluralin and 2-methyl-4-chlorophenoxyacetic acid sodium salt (MCPA-Na) on the susceptibility of the nocturnal moth Spodoptera litura to the insecticides λ-cyhalothrin, phoxim and bifenthrin. We found that larvae exposed to trifluralin or MCPA-Na became significantly less susceptible to both insecticides than non-exposed control larvae. Herbicide-treated larvae did not show altered growth under the used test conditions. However, heads of herbicide-treated larvae showed increased expression of the acetylcholinesterase genes SlAce1 and SlAce2. Moreover, the fat body and midgut of herbicide-treated larvae displayed elevated expression of detoxification genes (the carboxylesterase gene SlCarE; the glutathione S-transferase genes SlGSTe2 and SlGSTe3; the cytochrome P450 monooxygenase genes CYP6B48, CYP9A40 and CYP321B1). The CYP6B48 gene exhibited highest inducibility. In conclusion, the data of this study suggest that exposure of S. litura larvae to herbicides may stimulate detoxification mechanisms that compromise the efficacy of insecticides.