A tandem Kunitz protease inhibitor (KPI106)–serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula

Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza‐specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two‐hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.     

Nutrient demand and fungal access to resources control the carbon allocation to the symbiotic partners in tripartite interactions of Medicago truncatula

Legumes form tripartite interactions with arbuscular mycorrhizal fungi and rhizobia, and both root symbionts exchange nutrients against carbon from their host. The carbon costs of these interactions are substantial, but our current understanding of how the host controls its carbon allocation to individual root symbionts is limited. We examined nutrient uptake and carbon allocation in tripartite interactions of Medicago truncatula under different nutrient supply conditions, and when the fungal partner had access to nitrogen, and followed the gene expression of several plant transporters of the Sucrose Uptake Transporter (SUT) and Sugars Will Eventually be Exported Transporter (SWEET) family. Tripartite interactions led to synergistic growth responses and stimulated the phosphate and nitrogen uptake of the plant. Plant nutrient demand but also fungal access to nutrients played an important role for the carbon transport to different root symbionts, and the plant allocated more carbon to rhizobia under nitrogen demand, but more carbon to the fungal partner when nitrogen was available. These changes in carbon allocation were consistent with changes in the SUT and SWEET expression. Our study provides important insights into how the host plant controls its carbon allocation under different nutrient supply conditions and changes its carbon allocation to different root symbionts to maximize its symbiotic benefits.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

The Lotus japonicus acyl‐acyl carrier protein thioesterase FatM is required for mycorrhiza formation and lipid accumulation of Rhizophagus irregularis

Arbuscular mycorrhiza (AM) fungi establish symbiotic interactions with plants, providing the host plant with minerals, i.e. phosphate, in exchange for organic carbon. Arbuscular mycorrhiza fungi of the order Glomerales produce vesicles which store lipids as an energy and carbon source. Acyl‐acyl carrier protein (ACP) thioesterases (Fat) are essential components of the plant plastid‐localized fatty acid synthase and determine the chain length of de novo synthesized fatty acids. In addition to the ubiquitous FatA and FatB thioesterases, AM‐competent plants contain an additional, AM‐specific, FatM gene. Here, we characterize FatM from Lotus japonicus by phenotypically analyzing fatm mutant lines and by studying the biochemical function of the recombinant FatM protein. Reduced shoot phosphate content in fatm indicates compromised symbiotic phosphate uptake due to reduced arbuscule branching, and the fungus shows reduced lipid accumulation accompanied by the occurrence of smaller and less frequent vesicles. Lipid profiling reveals a decrease in mycorrhiza‐specific phospholipid forms, AM fungal signature fatty acids (e.g. 16:1ω5, 18:1ω7 and 20:3) and storage lipids. Recombinant FatM shows preference for palmitoyl (16:0)‐ACP, indicating that large amounts of 16:0 fatty acid are exported from the plastids of arbuscule‐containing cells. Stable isotope labeling with [¹³C₂]acetate showed reduced incorporation into mycorrhiza‐specific fatty acids in the fatm mutant. Therefore, colonized cells reprogram plastidial de novo fatty acid synthesis towards the production of extra amounts of 16:0, which is in agreement with previous results that fatty acid‐containing lipids are transported from the plant to the fungus.     

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae . The PAD2 gene encodes a γ-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1 . In contrast to the jasmonate-insensitive mutant coi1-1 , expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1 . Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.     

The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses

Abstract The rpoS (katF) gene, which encodes a RNA polymerase σ factor ( σ ^s ), regulates the virulence of Salmonella typhimurium in mice. In the present study, we show that rpoS mutants can be frequently found among laboratory strains of Salmonella . In addition, a rpoS mutation was identified in the S. typhi live oral vaccine Ty21a. Introduction of a wild-type rpoS gene in Ty21a allowed the bacteria to survive better under starvation conditions and increased their resistance to other stresses. These results contribute to a better understanding of the genetic background of the live typhoid oral vaccine Ty21a and suggest that the rpoS mutation may contribute to the safety of this strain in humans.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> constitutive photomorphogenic mutant, <Emphasis Type="Italic">bls1</Emphasis>,displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.     

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death

Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes.