John M. Labavitch

Transgenic expression of pear PGIP in tomato limits fungal colonization.

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

A Reevaluation of the Key Factors That Influence Tomato Fruit Softening and Integrity

The softening of fleshy fruits, such as tomato (Solanum lycopersicum), during ripening is generally reported to result principally from disassembly of the primary cell wall and middle lamella. However, unsuccessful attempts to prolong fruit firmness by suppressing the expression of a range of wall-modifying proteins in transgenic tomato fruits do not support such a simple model. ‘Delayed Fruit Deterioration’ (DFD) is a previously unreported tomato cultivar that provides a unique opportunity to assess the contribution of wall metabolism to fruit firmness, since DFD fruits exhibit minimal softening but undergo otherwise normal ripening, unlike all known nonsoftening tomato mutants reported to date. Wall disassembly, reduced intercellular adhesion, and the expression of genes associated with wall degradation were similar in DFD fruit and those of the normally softening ‘Ailsa Craig’. However, ripening DFD fruit showed minimal transpirational water loss and substantially elevated cellular turgor. This allowed an evaluation of the relative contribution and timing of wall disassembly and water loss to fruit softening, which suggested that both processes have a critical influence. Biochemical and biomechanical analyses identified several unusual features of DFD cuticles and the data indicate that, as with wall metabolism, changes in cuticle composition and architecture are an integral and regulated part of the ripening program. A model is proposed in which the cuticle affects the softening of intact tomato fruit both directly, by providing a physical support, and indirectly, by regulating water status.

Enzymatic hydrolysis of pretreated rice straw

California rice straw is being evaluated as a feedstock for production of power and fuel. This paper examines the initial steps in the process: pretreatment of rice straw and enzymatic hydrolysis of the polysaccharides in the pretreated material to soluble sugars. Rice straw was subjected to three distinct pretreatment procedures: acid-catalyzed steam explosion (Swan Biomass Company), acid hydrolysis (U.S. DOE National Renewable Energy Laboratory), and ammonia fiber explosion or AFEX (Texas A & M University). Standard conditions for each pretreatment were used, but none was optimized for rice straw specifically. Six commercial cellulases, products of Genencor International (USA), Novo (Denmark), Iogen (Canada) and Fermtech (Russia) were used for hydrolysis. The Swan- and the acid-pretreatments effectively removed hemicellulose from rice straw, providing high yields of fermentable sugars. The AFEX-pretreatment was distinctly different from other pretreatments in that it did not significantly solubilize hemicellulose. All three pretreatment procedures substantially increased enzymatic digestibility of rice straw. Three commercial Trichoderma-reesei-derived enzyme preparations: Cellulase 100L (Iogen), Spezyme CP (Genencor), and A1 (Fermtech), were more active on pretreated rice straw compared than others tested. Conditions for hydrolysis of rice straw using Cellulase 100L were evaluated. The supplementation of this enzyme preparation with cellobiase (Novozyme 188) significantly improved the parameters of hydrolysis for the Swan- and the acid-pretreated materials, but did not affect the hydrolysis of the AFEX-pretreated rice straw. The three pretreatment techniques were compared on a basis of a total yield and distribution of fermentable carbohydrates released by enzymatic hydrolysis (the highest possible substrate concentrations were used, 150 g/l for the Swan- and the acid- and 100 g/l for the AFEX-pretreated straw; enzyme loading of 6.7 filter paper units (FPU) and 6.7 cellobiase units (CBU) per gram of dry straw was the same for all pretreated materials). A combined yield of monosaccharides produced by a pretreatment step and by enzymatic hydrolysis was found to be 46, 42 and 37 g/l for the Swan-, the acid- and the AFEX-pretreated rice straw, respectively.

The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea

Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution.

Strangers in the matrix: plant cell walls and pathogen susceptibility

Early in infection, pathogens encounter the outer wall of plant cells. Because pathogen hydrolases targeting the plant cell wall are well-known components of virulence, it has been assumed that wall disassembly by the plant itself also contributes to susceptibility, and now this has been established experimentally. Understanding how plant morphological and developmental remodeling and pathogen cell wall targeted virulence influence infections provides new perspectives about plant-pathogen interactions. The plant cell wall can be an effective physical barrier to pathogens, but also it is a matrix where many proteins involved in pathogen perception are delivered. By breaching the wall, a pathogen potentially reveals itself to the plant and activates responses, setting off events that might halt or limit its advance.

Structure and expression of an inhibitor of fungal polygalacturonases from tomato

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.

Molecular Characterization of a Polygalacturonase Inhibitor from Pyrus communis L. cv Bartlett

A polygalacturonase inhibitor glycoprotein with an apparent molecular mass of 43 kD was purified from pear (Pyrus communis L. cv Bartlett) fruit. Chemical deglycosylation of this protein decreased the molecular mass to 34 kD. Gas chromatographic analysis suggests that N-linked glycosylation accounts for the majority of sugar moieties. Partial amino acid sequence analysis of the purified polygalacturonase inhibitor protein provided information used to amplify a corresponding cDNA by polymerase chain reactions. Multiple cloned products of these reactions were sequenced and the same open reading frame was identified in all of the products. It encodes a 36.5-kD polypeptide containing the amino acid sequences determined by protein sequencing and predicts a putative signal sequence of 24 amino acids and seven potential N-glycosylation sites. The expression of polygalacturonase inhibitor is regulated in a tissue-specific manner. Activity and mRNA level were much higher in fruit than in flowers or leaves.

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

Xylella fastidiosa is the causal agent of Pierces disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.

Ripening-Regulated Susceptibility of Tomato Fruit to Botrytis cinerea Requires NOR But Not RIN or Ethylene

Fruit ripening is a developmental process that is associated with increased susceptibility to the necrotrophic pathogen Botrytis cinerea. Histochemical observations demonstrate that unripe tomato (Solanum lycopersicum) fruit activate pathogen defense responses, but these responses are attenuated in ripe fruit infected by B. cinerea. Tomato fruit ripening is regulated independently and cooperatively by ethylene and transcription factors, including NON-RIPENING (NOR) and RIPENING-INHIBITOR (RIN). Mutations in NOR or RIN or interference with ethylene perception prevent fruit from ripening and, thereby, would be expected to influence susceptibility. We show, however, that the susceptibility of ripe fruit is dependent on NOR but not on RIN and only partially on ethylene perception, leading to the conclusion that not all of the pathways and events that constitute ripening render fruit susceptible. Additionally, on unripe fruit, B. cinerea induces the expression of genes also expressed as uninfected fruit ripen. Among the ripening-associated genes induced by B. cinerea are LePG (for polygalacturonase) and LeExp1 (for expansin), which encode cell wall-modifying proteins and have been shown to facilitate susceptibility. LePG and LeExp1 are induced only in susceptible rin fruit and not in resistant nor fruit. Thus, to infect fruit, B. cinerea relies on some of the processes and events that occur during ripening, and the fungus induces these pathways in unripe fruit, suggesting that the pathogen itself can initiate the induction of susceptibility by exploiting endogenous developmental programs. These results demonstrate the developmental plasticity of plant responses to the fungus and indicate how known regulators of fruit ripening participate in regulating ripening-associated pathogen susceptibility.