Fushi Wen

Effect of Pectin Methylesterase Gene Expression on Pea Root Development

Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the genes expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.

Extracellular Proteins in Pea Root Tip and Border Cell Exudates

Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% ± 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection.

Extracellular DNA Is Required for Root Tip Resistance to Fungal Infection

Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or “slime” composed of proteins, polysaccharides, and detached living cells called “border cells.” Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [32P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing 32P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) “stress” genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.

Identification and characterization of a rhizosphere β-galactosidase from Pisum sativum L.

Plant enzyme activities in the rhizosphere potentially are a resource for improved plant nutrition, soil fertility, bioremediation, and disease resistance. Here we report that a border cell specific β-galactosidase is secreted into the acidic extracellular environment surrounding root tips of pea, as well as bean, alfalfa, barrel medic, sorghum, and maize. No enzyme activity was detected in radish and Arabidopsis, species that do not produce viable border cells. The secreted enzyme activity was inhibited by galactose and 2-phenylethyl 1-thio-β-d-galactopyranoside (PETG) at concentrations that altered root growth without causing cell death. A tomato galactanase encoding gene was used as a probe to isolate a full length pea cDNA clone (BRDgal1) from a root cap-border cell cDNA library. Southern blot analysis using full length BRDgal1 as a probe revealed 1–2 related sequences within the pea genome. BRDgal1 mRNA expression was analysed by whole mount in situ hybridization (WISH) and found to occur in the outermost peripheral layer of the cap and in suspensions of detached border cells. No expression was detected within the body of the root cap. Repeated efforts to develop viable hairy root clones expressing BRDgal1 antisense mRNA under the control of the CaMV35S promoter, whose expression in the root cap is limited to cells at the root cap periphery only during root emergence, were unsuccessful. These data suggest that altered expression of this enzyme is deleterious to early root development.

Lethality of inducible, meristem-localized ectopic β-glucuronidase expression in plants

GUSA fromEscherichia coli, encoded by theuidA gene, has been successfully used as a plant reporter system for more than a decade with no reported deleterious effects. However, when expressed in coordination with a UDP-glucuronosyltransferase isolated from the root cap meristem ofPisum sativum (PsUGT1) at the onset of mitosis, GUSA expression was lethal in pea, alfalfa, andArabidopsis thaliana. These unexpected results indicate that, under some circumstances, using GUSA in plants is incompatible with life and suggest that the cell-specific lethal phenotype might be useful in selecting for genes specifically involved in regulating the G2-M phase of the cell cycle.

Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50–60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.

Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. (rcpme1) and its use in the study of gene activity

A genomic clone of a pea pectinmethylesterase encoding gene, rcpme1, was isolated; the promoter region was found to include regions of homology to phenylalanine ammonia lyase (PAL) and nodulin gene promoters. Agrobacterium rhizogenes mediated hairy roots were used for rcpme1 expression and functional analysis in pea. Patterns of rcpme1 expression in cultured hairy roots, measured using uidA encoding β-glucuronidase (GUS) as a reporter gene, were distinct from patterns which occur in normal pea roots. No reporter gene expression occurred in transgenic Arabidopsis thaliana, whose roots do not produce border cells. Border cell number from transgenic hairy roots expressing rcpme1 anti-sense mRNA under the control of its 2.75 kb 5′ flanking sequence was reduced by > 50%. Nodulation genes of Rhizobium leguminosarum were used as a marker to document that roots with reduced production of border cells and other root cap exudates have a corresponding reduction in levels of biologically active signal molecules. Direct measurements were used to confirm that most of the exudate harvested from young, unwounded roots of normal pea plants is derived from the root tip region where rcpme1 is expressed. The potential application of the rcpme1 gene as a molecular marker for root exudate production is discussed.

Visualization of extracellular DNA released during border cell separation from the root cap

PREMISE OF THE STUDY Root border cells are programmed to separate from the root cap as it penetrates the soil environment, where the cells actively secrete >100 extracellular proteins into the surrounding mucilage. The detached cells function in defense of the root tip by an extracellular trapping process that also requires DNA, as in mammalian white blood cells. Trapping in animals and plants is reversed by treatment with DNase, which results in increased infection. The goal of this study was to evaluate the role of DNA in the structural integrity of extracellular structures released as border cells disperse from the root tip upon contact with water. METHODS DNA stains including crystal violet, toluidine blue, Hoechst 33342, DAPI, and SYTOX green were added to root tips to visualize the extracellular mucilage as it absorbed water and border cell populations dispersed. DNase I was used to assess structural changes occurring when extracellular DNA was degraded. KEY RESULTS Complex masses associated with living border cells were immediately evident in response to each stain, including those that are specific for DNA. Treating with DNase I dramatically altered the appearance of the extracellular structures and their association with border cells. No extracellular DNA was found in association with border cells killed by freezing or high-speed centrifugation. This observation is consistent with the hypothesis that, as with border cell extracellular proteins, DNA is secreted by living cells. CONCLUSION DNA is an integral component of border cell extracellular traps.

Altered growth and root tip morphology in Pisum sativum L. in response to altered expression of a gene expressed in border cells

Background and aimsRecent studies suggest that root border cells function in defense of the root by an extracellular DNA-based trapping mechanism similar to that described in mammalian white blood cells. Genes controlling the specialized properties of border cells as they detach from the root tip therefore are of interest.MethodsmRNA differential display was used to identify Brd13, a sequence expressed in border cells but not other root tissues. RNase protection and mRNA Northern blot analyses, and reporter gene expression under the control of the Brd13 promoter in transgenic hairy roots were used to confirm localized expression. Phenotype analysis of transgenic hairy roots expressing Brd13 antisense mRNA was carried out.ResultsBrd13 was expressed constitutively in border cells but not in leaves, stems, or roots without border cells. The predicted protein shares sequence similarity with flavin-binding proteins. Transgenic hairy roots expressing Brd13 antisense mRNA exhibited abnormal growth and morphology.ConclusionsWe report here that altered expression of a putative flavin-binding protein in border cells resulted in altered root development. Flavin-binding proteins play key roles in development, defense, and local auxin biosynthesis. The Brd13 gene and its promoter may be useful in creating defined changes in root development and defense.