Wall Appositions Research Articles

The development of pleiotropic phenotypes in powdery mildew-resistant barley and Arabidopsis thaliana mlo mutants is linked to nitrogen availability.

Powdery mildew-resistant barley (Hordeum vulgare) and Arabidopsis thaliana mlo mutant plants exhibit pleiotropic phenotypes such as the spontaneous formation of callose-rich cell wall appositions and early leaf chlorosis and necrosis, indicative of premature leaf senescence. The exogenous factors governing the occurrence of these undesired side effects remain poorly understood. Here, we characterised the formation of these symptoms in detail. Ultrastructural analysis revealed that the callose-rich cell wall depositions spontaneously formed in A. thaliana mlo mutants are indistinguishable from those induced by the bacterial pattern epitope, flagellin 22 (flg22). We further found that increased plant densities during culturing enhance the extent of the leaf senescence syndrome in A. thaliana mlo mutants. Application of a liquid fertiliser rescued the occurrence of leaf chlorosis and necrosis in both A. thaliana and barley mlo mutant plants. Controlled fertilisation experiments uncovered nitrogen as the macronutrient whose deficiency promotes the extent of pleiotropic phenotypes in A. thaliana mlo mutants. Light intensity and temperature had a modulatory impact on the incidence of leaf necrosis in the case of barley mlo mutant plants. Collectively, our data indicate that the development of pleiotropic phenotypes associated with mlo mutants is governed by various exogenous factors.

A GH81-type β-glucan-binding protein enhances colonization by mutualistic fungi in barley

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of β-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated β-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a β-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S.vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley-adapted pathogens.

Iron redistribution induces oxidative burst and resistance in maize against Curvularia lunata.

ΔClnps6 induced iron redistribution in maize B73 leaf cells and resulted in reactive oxygen species (ROS) burst to enhance plant resistance against Curvularia lunata. Iron is an indispensable co-factor of various crucial enzymes that are involved in cellular metabolic processes and energy metabolism in eukaryotes. For this reason, plants and pathogens compete for iron to maintain their iron homeostasis, respectively. In our previous study, ΔClnps6, the extracellular siderophore biosynthesis deletion mutant of Curvularia lunata, was sensitive to exogenous hydrogen peroxide and virulence reduction. However, the mechanism was not studied. Here, we report that maize B73 displayed highly resistance to ΔClnps6. The plants recruited more iron at cell wall appositions (CWAs) to cause ROS bursts. Intracellular iron deficiency induced by iron redistribution originated form up-regulated expression of genes involved in intracellular iron consumption in leaves and absorption in roots. The RNA-sequencing data also showed that the expression of respiratory burst oxidase homologue (ZmRBOH4) and NADP-dependent malic enzyme 4 (ZmNADP-ME4) involved in ROS production was up-regulated in maize B73 after ΔClnps6 infection. Simultaneously, jasmonic acid (JA) biosynthesis genes lipoxygenase (ZmLOX), allene oxide synthase (ZmAOS), GA degradation gene gibberellin 2-beta-dioxygenase (ZmGA2OX6) and ABA degradation genes abscisic acid hydroxylase (ZmABH1, ZmABH2) involved in iron homeostasis were up-regulated expression. Ferritin1 (ZmFER1) positive regulated maize resistance against C. lunata via ROS burst under Fe-limiting conditions. Overall, our results showed that iron played vital roles in activating maize resistance in B73-C. lunata interaction.

Adult resistance genes to barley powdery mildew confer basal penetration resistance associated with broad-spectrum resistance.

Powdery mildew isa major disease of barley (Hordeum vulgare L.) for which breeders have traditionally relied on dominant, pathogen race-specific resistance genes for genetic control. Directional selection pressures in extensive monocultures invariably result in such genes being overcome as the pathogen mutates to evade recognition. This has led to a widespread reliance on fungicides and a single broad-spectrum recessive resistance provided by the mlo gene. The range of resistance genes and alleles found in wild crop relatives and landraces has been reduced in agricultural cultivars through an erosion of genetic diversity during domestication and selective breeding. Three novel major-effect adult plant resistance (APR) genes from landraces, designated Resistance to Blumeria graminis f. sp. hordei (Rbgh1 to Rbgh3), were identified in the terminal regions of barley chromosomes 5HL, 7HS, and 1HS, respectively. The phenotype of the new APR genes showed neither pronounced penetration resistance, nor the spontaneous necrosis and mesophyll cell death typical of mlo resistance, nor a whole epidermal cell hypersensitive response, typical of race-specific resistance. Instead, resistance was localized to the site of attempted penetration in an epidermal cell and was associated with cell wall appositions and cytosolic vesicle-like bodies, and lacked strong induction of reactive oxygen species. The APR genes exhibited differences in vesicle-like body sizes, their distribution, and the extent of localized 3,3-diaminobenzidine staining in individual doubled haploid lines. The results revealed a set of unique basal penetration resistance genes that offer opportunities for combining different resistance mechanisms in breeding programs for robust mildew resistance.

The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

VAPYRIN attenuates defence by repressing PR gene induction and localized lignin accumulation during arbuscular mycorrhizal symbiosis of Petunia hybrida.

Summary The intimate association of host and fungus in arbuscular mycorrhizal (AM) symbiosis can potentially trigger induction of host defence mechanisms against the fungus, implying that successful symbiosis requires suppression of defence.We addressed this phenomenon by using AM‐defective vapyrin (vpy) mutants in Petunia hybrida, including a new allele (vpy‐3) with a transposon insertion close to the ATG start codon. We explore whether abortion of fungal infection in vpy mutants is associated with the induction of defence markers, such as cell wall alterations, accumulation of reactive oxygen species (ROS), defence hormones and induction of pathogenesis‐related (PR) genes.We show that vpy mutants exhibit a strong resistance against intracellular colonization, which is associated with the generation of cell wall appositions (papillae) with lignin impregnation at fungal entry sites, while no accumulation of defence hormones, ROS or callose was observed. Systematic analysis of PR gene expression revealed that several PR genes are induced in mycorrhizal roots of the wild‐type, and even more in vpy plants. Some PR genes are induced exclusively in vpy mutants.Our results suggest that VPY is involved in avoiding or suppressing the induction of a cellular defence syndrome that involves localized lignin deposition and PR gene induction.

Prior exposure of Arabidopsis seedlings to mechanical stress heightens jasmonic acid-mediated defense against necrotrophic pathogens

BackgroundProlonged mechanical stress (MS) causes thigmomorphogenesis, a stress acclimation response associated with increased disease resistance. What remains unclear is if; 1) plants pre-exposed to a short period of repetitive MS can prime defence responses upon subsequent challenge with necrotrophic pathogens, 2) MS mediates plant immunity via jasmonic acid (JA) signalling, and 3) a short period of repetitive MS can cause long-term changes in gene expression resembling a stress-induced memory. To address these points, 10-days old juvenile Arabidopsis seedlings were mechanically stressed for 7-days using a soft brush and subsequently challenged with the necrotrophic pathogens, Alternaria brassicicola, and Botrytis cinerea. Here we assessed how MS impacted structural cell wall appositions, disease symptoms and altered gene expression in response to infection.ResultsThe MS-treated plants exhibited enhanced cell wall appositions and jasmonic acid (JA) accumulation that correlated with a reduction in disease progression compared to unstressed plants. The expression of genes involved in JA signalling, callose deposition, peroxidase and phytoalexin biosynthesis and reactive oxygen species detoxification were hyper-induced 4-days post-infection in MS-treated plants. The loss-of-function in JA signalling mediated by the JA-insensitive coronatine-insensitive 1 (coi1) mutant impaired the hyper-induction of defense gene expression and promoted pathogen proliferation in MS-treated plants subject to infection. The basal expression level of PATHOGENESIS-RELATED GENE 1 and PLANT DEFENSIN 1.2 defense marker genes were constitutively upregulated in rosette leaves for 5-days post-MS, as well as in naïve cauline leaves that differentiated from the inflorescence meristem well after ceasing MS.ConclusionThis study reveals that exposure of juvenile Arabidopsis plants to a short repetitive period of MS can alter gene expression and prime plant resistance upon subsequent challenge with necrotrophic pathogens via the JA-mediated COI1 signalling pathway. MS may facilitate a stress-induced memory to modulate the plant’s response to future stress encounters. These data advance our understanding of how MS primes plant immunity against necrotrophic pathogens and how that could be utilised in sustainable agricultural practices.

A secreted fungal histidine‐ and alanine‐rich protein regulates metal ion homeostasis and oxidative stress

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S.indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

Visualization of the halo region in plant–powdery mildew interactions by cryoscanning electron microscopy

Halos are circular or ring-shaped cell wall appositions surrounding pathogen entry sites on host leaves. The diameter of halos on infected Blumeria graminis wheat leaves varied between 10 and 100 μm, which reflected specificity of local host plant responses. Using cryoscanning electron microscopy (cryoSEM) without metal sputtering we observed halos as light circles or ring-shaped patterns on the leaves of cereals infected with B. graminis, and on the leaves of several dicotyledonous plants infected with specialized powdery mildews. Hydrogen peroxide treatment of wheat leaves increased the number of cryoSEM detected halos. They were more distinct than in the control without treatment and showed better contrast with surrounding epidermis. The presented cryoSEM method allows simultaneous monitoring of halos and fungal structures and, possibly, visualizes penetration sites with elevated production of reactive oxygen species.

The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants.

Summary Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The lignification of cell wall appositions is a conserved basal defence mechanism in the plant innate immune response. However, the function of laccase in defence‐induced lignification has not been described. Screening of an SSH library of a resistant cotton cultivar, Jimian20, inoculated with V. dahliae revealed a laccase gene that was strongly induced by the pathogen. This gene was phylogenetically related to AtLAC15 and contained domains conserved by laccases; therefore, we named it GhLAC15. Quantitative reverse transcription‐polymerase chain reaction indicated that GhLAC15 maintained higher expression levels in tolerant than in susceptible cultivars. Overexpression of GhLAC15 enhanced cell wall lignification, resulting in increased total lignin, G monolignol and G/S ratio, which significantly improved the Verticillium wilt resistance of transgenic Arabidopsis. In addition, the levels of arabinose and xylose were higher in transgenic plants than in wild‐type plants, which resulted in transgenic Arabidopsis plants being less easily hydrolysed. Furthermore, suppression of the transcriptional level of GhLAC15 resulted in an increase in susceptibility in cotton. The content of monolignol and the G/S ratio were lower in silenced cotton plants, which led to resistant cotton cv. Jimian20 becoming susceptible. These results demonstrate that GhLAC15 enhances Verticillium wilt resistance via an increase in defence‐induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum. This study broadens our knowledge of defence‐induced lignification and cell wall modifications as defence mechanisms against V. dahliae.

An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants

SummaryCell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.

SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis.

Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops.

Biochemical and cell wall ultrastructural changes in crown tissue of banana (Musa AAA ‘Berangan’) fruit as mediated by UVC irradiation against crown rot fungal infection

The reduction of crown rot disease observed in UVC-irradiated banana fruit, ‘Berangan’ was associated with activation of the defense response mechanisms, increased peroxidase and polyphenol oxidase activities as well as accumulation of lignin and phenolic compounds in its crown tissue, but not phenylalanine ammonia-lyase activity. Transmission electron microscope micrographs revealed that 0.01kJm−2 UVC retarded the losses of cell compartments and disintegrations of fibril fraction in cell wall structure of the crown tissue. UVC treatment 24h after fungal inoculation induced the accumulation of phenol-storing cells that could create an antimicrobial environment in the host tissue for combating the fungal colonization. Moreover, formation of cell wall lignification and heterogenous matrix of cell wall appositions at site of fungal penetration was clearly observed in UVC-irradiated crown tissue, which otherwise absent in the control crowns. These structures might be important physical barriers that were induced by the irradiation to prevent diffusions of toxin and enzyme from the crown rot-infecting fungi.

Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis.

Serine palmitoyltransferase (SPT), a pyridoxyl-5'-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H2O2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants.

Tempered mlo broad-spectrum resistance to barley powdery mildew in an Ethiopian landrace

Recessive mutations in the Mlo gene confer broad spectrum resistance in barley (Hordeum vulgare) to powdery mildew (Blumeria graminis f. sp. hordei), a widespread and damaging disease. However, all alleles discovered to date also display deleterious pleiotropic effects, including the naturally occurring mlo-11 mutant which is widely deployed in Europe. Recessive resistance was discovered in Eth295, an Ethiopian landrace, which was developmentally controlled and quantitative without spontaneous cell wall appositions or extensive necrosis and loss of photosynthetic tissue. This resistance is determined by two copies of the mlo-11 repeat units, that occur upstream to the wild-type Mlo gene, compared to 11–12 in commonly grown cultivars and was designated mlo-11 (cnv2). mlo-11 repeat unit copy number-dependent DNA methylation corresponded with cytological and macroscopic phenotypic differences between copy number variants. Sequence data indicated mlo-11 (cnv2) formed via recombination between progenitor mlo-11 repeat units and the 3′ end of an adjacent stowaway MITE containing region. mlo-11 (cnv2) is the only example of a moderated mlo variant discovered to date and may have arisen by natural selection against the deleterious effects of the progenitor mlo-11 repeat unit configuration.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

SummaryVarieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking‐out susceptibility S‐genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S‐genes of phylogenetic clade V up‐regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock‐down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock‐down of MdMLO19 reduced PM disease severity by 75%, whereas the knock‐down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM‐resistant and PM‐susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock‐down induces a very significant level of resistance.

Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion.

완두콩(Pisum sativum L.) 상처에서 유도되는 불용성 산성 인버타제의 면역조직화

Invertase, that hydrolyzes sucrose into glucose and fructose, plays a great role in carbohydrate reallocation between the photosynthetic source tissue and various sink tissues. Invertase also occurs in a variety of isoforms for various functions in plants. Insoluble invertases were extracted only in buffer solutions containing high concentrations of salt. Within these classes, acid invertase has an optimum activity at acidic pH (pH 4-5). Induction of insoluble acid invertase (INAC-INV) in leaf, stem, and root tissues in response to physical wounding has been investigated. To detect the localization of INAC-INV within the plant, immunolocalization has been performed. In this study, the accumulation of INAC-INV was noticeable to reach maximum levels on 72 hr after mechanical injuries. INAC-INV was induced in wounded leaves 3 times more than control leaves. Immunolocalization results showed that INAC-INV accumulated in wall appositions and intercellular spaces. INAC-INV was also localized at sieve cell walls in phloem tissues close to the site of wounding. Taken together, this study suggested that INAC-INV induction upon wounding injuries can play a role on responses to the high energy demand for wound healing process.

Monitoring of cell wall modifications during callogenesis in Stylosanthes guianensis (Fabaceae) under salt stress conditions

Callogenesis was induced from Stylosanthes guianensis (Aubl.) Sw. cv. CIAT-184 hypocotyl explants cultured in the presence of different concentrations of NaCl. Whereas calluses formed at 50 mM NaCl did not show significant changes, concentrations in the range of 100-250 mM NaCl influenced callus formation and sur- vival. The concentrations which reduced the callus for- mation and survival by 50 % with respect to the controls (I50) were 190 and 225 mM NaCl, respectively. Callus growth was also affected being 160 mM NaCl the con- centration that reduced the fresh weight gain in 50 %, in comparison to the control. The cell walls of calluses formed at higher NaCl concentrations showed a lower cellulose content, which was accompanied by an increase in protein, phenolics, and esters, as revealed by Fourier transform infrared spectroscopy. These overall cell wall modifications were accompanied by the formation of cell wall appositions enriched with homogalacturonan and rhamnogalacturonan components, and also with an increase of arabinogalactan proteins in cell walls. These results evidence the ability of cells to adjust the cell wall com- position under salt stress conditions, as part of the strategy to sustain their growth in such environment.