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.

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.

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.

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.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.

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.

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.

Biochemical properties and in planta effects of NopM, a rhizobial E3 ubiquitin ligase.

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

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.

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.

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.

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.

The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation.

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.

Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti under free-living and symbiotic oxidative stress.

The development of legume nitrogen-fixing nodules is regulated by reactive oxygen species (ROS) produced by symbionts. Several regulators from Rhizobium are involved in ROS sensing. In a previous study, we found that Sinorhizobium meliloti LsrB regulates lipopolysaccharide production and is associated with H2 O2 accumulation in alfalfa (Medicago sativa) nodules. However, its underlying regulatory mechanism remains unclear. Here, we report that the cysteine residues in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa nodulation and nitrogen fixation. Moreover, LsrB directly activated the transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase, responsible for glutathione synthesis) and this regulation required the cysteine (Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense oxidative stress via the formation of intermolecular disulfide bonds, generating LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive regulatory site for the induction of downstream genes, whereas C146 and C275 play negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions displayed altered phenotypes in respect to their adaptation to oxidative stress, nodulation and nitrogen fixation-related plant growth. Our findings demonstrate that S. meliloti LsrB modulates alfalfa nodule development by directly regulating downstream gene expression via a post-translational strategy.

Identification of Two Cytochrome Monooxygenase P450 Genes, CYP321A7 and CYP321A9, from the Tobacco Cutworm Moth (Spodoptera Litura) and Their Expression in Response to Plant Allelochemicals.

Larvae of the polyphagous tobacco cutworm moth, Spodoptera litura (S. litura), encounter potentially toxic allelochemicals in food. It is therefore important for S. litura to produce detoxification enzymes such as cytochrome P450 monooxygenases (P450s). In this study, we have identified two novel cytochrome P450 genes of S. litura, named CYP321A7 and CYP321A9. Phylogenetic analysis indicated that they belong to the CYP321A subfamily. Expression levels of these genes at different development stages were determined by real-time quantitative polymerase chain reaction (PCR). The highest expression was found in the midgut and the fat body. Larvae fed with a diet supplemented with xanthotoxin or coumarin showed a strongly increased expression of CYP321A7 and CYP321A9 in the midgut and fat body as compared to larvae that consumed a control diet. In contrast, larvae consuming a diet containing aflatoxin B1 or quercetin did not induce the expression of these genes. CYP321A7 and CYP321A9 showed different expression profiles with respect to certain allelochemicals. For example, a diet containing cinnamic acid stimulated the expression of CYP321A9, whereas no changes were observed for CYP321A7. We suggest that the fine tuning of P450 gene expression is an important adaptation mechanism that allows polyphagous S. litura larvae to survive in a changing chemical environment.

The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate.

Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.

Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research.

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor-induced defense signaling in Arabidopsis thaliana and chitin-induced nodulation signaling in Lotus japonicus.

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1-AtCERK1 and LjNFR5-AtCERK1, were expressed in cerk1-2, an A. thaliana CERK1 mutant lacking chitin-induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1-2 expressing both chimeras accumulated reactive oxygen species, expressed chitin-responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor-binding protein), respectively. The chimeras, named OsCERK1-LjNFR1 and OsCEBiP-LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1-1; nfr5-2) carrying a GUS (ß-glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF-induced defense signaling in non-legumes and chitin-induced nodulation signaling in legumes.

Expression analysis of two P450 monooxygenase genes of the tobacco cutworm moth (Spodoptera litura) at different developmental stages and in response to plant allelochemicals.

Cytochrome P450 monooxygenases (P450s) of insects are known to be involved in the metabolism or detoxification of plant allelochemicals and insecticides. Spodoptera litura (Lepidoptera, Noctuidae) is a polyphagous moth responsible for severe yield losses in many crops. In this study, two full-length P450 genes, CYP6B48 and CYP6B58, were cloned from S. litura. The cDNA sequences encode proteins with 503 and 504 amino acids, respectively. Phylogenetic analysis revealed that CYP6B48 and CYP6B58 belong to the CYP6B subfamily of P450s. Quantitative real-time PCR analyses showed that CYP6B48 and CYP6B58 were expressed only at larval stage, but not at pupal and adult stages. The highest levels of transcripts were found in the midguts and fat bodies of the larvae. No expression was detected in the ovary or hemolymph. Feeding with diets containing cinnamic acid, quercetin, or coumarin did not affect expression of CYP6B48. In contrast, diet supplemented with xanthotoxin dramatically increased the levels of CYP6B48 transcript in the midgut and fat bodies. Larvae fed with flavone had high levels of transcript of CYP6B48 in the midgut, whereas only slightly elevated levels were found in the fat bodies. Effects of the tested allelochemicals on CYP6B58 expression were minor. Hence, our findings show that S. litura responds to specific allelochemicals such as xanthotoxin with the accumulation of CYP6B48 transcripts, suggesting that specific signals in the food control the insect's ability to convert toxic allelochemicals to less harmful forms at the transcriptional level.