Cellulose synthase-like D movement in the plasma membrane requires enzymatic activity.

Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate ß-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis. Actin is required to target CSLD to cell tips concomitant with cell expansion, but not to cell plates, which depend on actin and CSLD for structural support. Like Cellulose Synthase (CESA), CSLD requires catalytic activity to move in the plasma membrane. We discovered that CSLD moves significantly faster, with shorter duration and less linear trajectories than CESA. In contrast to CESA, CSLD movement was insensitive to the cellulose synthesis inhibitor isoxaben, suggesting that CSLD and CESA function within different complexes possibly producing structurally distinct cellulose microfibrils.

Cellulose synthesis complexes are homo-oligomeric and hetero-oligomeric in Physcomitrium patens.

The common ancestor of seed plants and mosses contained homo-oligomeric cellulose synthesis complexes (CSCs) composed of identical subunits encoded by a single CELLULOSE SYNTHASE (CESA) gene. Seed plants use different CESA isoforms for primary and secondary cell wall deposition. Both primary and secondary CESAs form hetero-oligomeric CSCs that assemble and function in planta only when all the required isoforms are present. The moss Physcomitrium (Physcomitrella) patens has seven CESA genes that can be grouped into two functionally and phylogenetically distinct classes. Previously, we showed that PpCESA3 and/or PpCESA8 (class A) together with PpCESA6 and/or PpCESA7 (class B) form obligate hetero-oligomeric complexes required for normal secondary cell wall deposition. Here, we show that gametophore morphogenesis requires a member of class A, PpCESA5, and is sustained in the absence of other PpCESA isoforms. PpCESA5 also differs from the other class A PpCESAs as it is able to self-interact and does not co-immunoprecipitate with other PpCESA isoforms. These results are consistent with the hypothesis that homo-oligomeric CSCs containing only PpCESA5 subunits synthesize cellulose required for gametophore morphogenesis. Analysis of mutant phenotypes also revealed that, like secondary cell wall deposition, normal protonemal tip growth requires class B isoforms (PpCESA4 or PpCESA10), along with a class A partner (PpCESA3, PpCESA5, or PpCESA8). Thus, P. patens contains both homo-oligomeric and hetero-oligomeric CSCs.

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms.

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Preferred crystallographic orientation of cellulose in plant primary cell walls.

Cellulose, the most abundant biopolymer on earth, is a versatile, energy rich material found in the cell walls of plants, bacteria, algae, and tunicates. It is well established that cellulose is crystalline, although the orientational order of cellulose crystallites normal to the plane of the cell wall has not been characterized. A preferred orientational alignment of cellulose crystals could be an important determinant of the mechanical properties of the cell wall and of cellulose-cellulose and cellulose-matrix interactions. Here, the crystalline structures of cellulose in primary cell walls of onion (Allium cepa), the model eudicot Arabidopsis (Arabidopsis thaliana), and moss (Physcomitrella patens) were examined through grazing incidence wide angle X-ray scattering (GIWAXS). We find that GIWAXS can decouple diffraction from cellulose and epicuticular wax crystals in cell walls. Pole figures constructed from a combination of GIWAXS and X-ray rocking scans reveal that cellulose crystals have a preferred crystallographic orientation with the (200) and (110)/([Formula: see text]) planes preferentially stacked parallel to the cell wall. This orientational ordering of cellulose crystals, termed texturing in materials science, represents a previously unreported measure of cellulose organization and contradicts the predominant hypothesis of twisting of microfibrils in plant primary cell walls.

Knocking Out the Wall: Revised Protocols for Gene Targeting in Physcomitrella patens.

The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analysing the genotypes and phenotypes of the resulting transformed lines.

Functional Characterization of a Glycosyltransferase from the Moss Physcomitrella patens Involved in the Biosynthesis of a Novel Cell Wall Arabinoglucan.

Mixed-linkage (1,3;1,4)-ß-glucan (MLG), an abundant cell wall polysaccharide in the Poaceae, has been detected in ascomycetes, algae, and seedless vascular plants, but not in eudicots. Although MLG has not been reported in bryophytes, a predicted glycosyltransferase from the moss Physcomitrella patens (Pp3c12_24670) is similar to a bona fide ascomycete MLG synthase. We tested whether Pp3c12_24670 encodes an MLG synthase by expressing it in wild tobacco (Nicotiana benthamiana) and testing for release of diagnostic oligosaccharides from the cell walls by either lichenase or (1,4)-ß-glucan endohydrolase. Lichenase, an MLG-specific endohydrolase, showed no activity against cell walls from transformed N. benthamiana, but (1,4)-ß-glucan endohydrolase released oligosaccharides that were distinct from oligosaccharides released from MLG by this enzyme. Further analysis revealed that these oligosaccharides were derived from a novel unbranched, unsubstituted arabinoglucan (AGlc) polysaccharide. We identified sequences similar to the P. patens AGlc synthase from algae, bryophytes, lycophytes, and monilophytes, raising the possibility that other early divergent plants synthesize AGlc. Similarity of P. patens AGlc synthase to MLG synthases from ascomycetes, but not those from Poaceae, suggests that AGlc and MLG have a common evolutionary history that includes loss in seed plants, followed by a more recent independent origin of MLG within the monocots.

Cellulose synthase 'class specific regions' are intrinsically disordered and functionally undifferentiated.

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function.

Functional Specialization of Cellulose Synthase Isoforms in a Moss Shows Parallels with Seed Plants.

The secondary cell walls of tracheary elements and fibers are rich in cellulose microfibrils that are helically oriented and laterally aggregated. Support cells within the leaf midribs of mosses deposit cellulose-rich secondary cell walls, but their biosynthesis and microfibril organization have not been examined. Although the Cellulose Synthase (CESA) gene families of mosses and seed plants diversified independently, CESA knockout analysis in the moss Physcomitrella patens revealed parallels with Arabidopsis (Arabidopsis thaliana) in CESA functional specialization, with roles for both subfunctionalization and neofunctionalization. The similarities include regulatory uncoupling of the CESAs that synthesize primary and secondary cell walls, a requirement for two or more functionally distinct CESA isoforms for secondary cell wall synthesis, interchangeability of some primary and secondary CESAs, and some CESA redundancy. The cellulose-deficient midribs of ppcesa3/8 knockouts provided negative controls for the structural characterization of stereid secondary cell walls in wild type P. patens Sum frequency generation spectra collected from midribs were consistent with cellulose microfibril aggregation, and polarization microscopy revealed helical microfibril orientation only in wild type leaves. Thus, stereid secondary walls are structurally distinct from primary cell walls, and they share structural characteristics with the secondary walls of tracheary elements and fibers. We propose a mechanism for the convergent evolution of secondary walls in which the deposition of aggregated and helically oriented microfibrils is coupled to rapid and highly localized cellulose synthesis enabled by regulatory uncoupling from primary wall synthesis.

The valine and lysine residues in the conserved FxVTxK motif are important for the function of phylogenetically distant plant cellulose synthases.

Cellulose synthases (CESAs) synthesize the ß-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA.

A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae).

PREMISE OF THE STUDY: A method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. METHODS AND RESULTS: A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. CONCLUSIONS: Compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

Virulence of Xanthomonas translucens pv. poae Isolated from Poa annua.

Bacterial wilt is a vascular wilt disease caused by Xanthomonas translucens pv. poae that infects Poa annua, a grass that is commonly found on golf course greens throughout the world. Bacterial wilt causes symptoms of etiolation, wilting, and foliar necrosis. The damage is most prevalent during the summer and the pathogen can kill turf under conditions optimal for disease development. Fifteen isolates of X. translucens pv. poae were collected from northern regions in the United States and tested for virulence against P. annua. All 15 isolates were pathogenic on P. annua, but demonstrated variable levels of virulence when inoculated onto P. annua under greenhouse conditions. The isolates were divided into two virulence groups. The first group containing four isolates generally resulted in less than 40% mortality following inoculation. The second group, containing the other eleven isolates, produced between 90 and 100% mortality following inoculation. These results suggest that differences in the virulence of bacterial populations present on a golf course may result in more or less severe amounts of observed disease.

Identification, Characterization, and Distribution of Acidovorax avenae subsp. avenae Associated with Creeping Bentgrass Etiolation and Decline.

Bacterial etiolation and decline caused by Acidovorax avenae subsp. avenae is an emerging disease of creeping bentgrass (Agrostis stolonifera) in and around the transition zone, a unique area of turfgrass culture between cool and warm regions of the United States. It is suspected that the disease has been present for many years, although diagnosis of the first occurrence was not reported until 2010. Solicitation of samples from golf courses in 2010 and 2011 was undertaken to investigate the prevalence and dissemination of Acidovorax avenae subsp. avenae on creeping bentgrass. At least 21 isolates from 13 states associated with these outbreaks on golf courses were confirmed as A. avenae subsp. avenae by pathogenicity assays and 16S rDNA sequence analysis at two independent locations. Pathogenicity testing of bacterial isolates from creeping bentgrass samples exhibiting heavy bacterial streaming confirmed A. avenae subsp. avenae as the only bacterium to cause significant disease symptoms and turfgrass decline. Host range inoculations revealed isolates of A. avenae subsp. avenae to be pathogenic on all Agrostis stolonifera cultivars tested, with slight but significant differences in disease severity on particular cultivars. Other turfgrass hosts tested were only mildly susceptible to Acidovorax avenae subsp. avenae infection. This study initiated research on A. avenae subsp. avenae pathogenicity causing a previously uncharacterized disease of creeping bentgrass putting greens in the United States.

Variation in plant defense against invasive herbivores: evidence for a hypersensitive response in eastern hemlocks (Tsuga canadensis).

Herbivores can trigger a wide array of morphological and chemical changes in their host plants. Feeding by some insects induces a defensive hypersensitive response, a defense mechanism consisting of elevated H(2)O(2) levels and tissue death at the site of herbivore feeding. The invasive hemlock woolly adelgid Adelges tsugae ('HWA') and elongate hemlock scale Fiorinia externa ('EHS') feed on eastern hemlocks; although both are sessile sap feeders, HWA causes more damage than EHS. The rapid rate of tree death following HWA infestation has led to the suggestion that feeding induces a hypersensitive response in hemlock trees. We assessed the potential for an herbivore-induced hypersensitive response in eastern hemlocks by measuring H(2)O(2) levels in foliage from HWA-infested, EHS-infested, and uninfested trees. Needles with settled HWA or EHS had higher H(2)O(2) levels than control needles, suggesting a localized hypersensitive plant response. Needles with no direct contact to settled HWA also had high H(2)O(2) levels, suggesting that HWA infestation may induce a systemic defense response in eastern hemlocks. There was no similar systemic defensive response in the EHS treatment. Our results showed that two herbivores in the same feeding guild had dramatically different outcomes on the health of their shared host.