Paul H. Goodwin

Recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi

Both saprophytic and plant parasitic fungi produce extracellular enzymes which can degrade the cell wall components of plants. These fungi not only digest plant cell wall polymers to obtain an important nutrient source but also degrade the cell wall to aid in penetrating cells and spreading through plant tissue. DeBary (1886) was the first to suggest that extracellular enzymes may be involved in the infection process of plant pathogenic fungi. Since then, much research has been focused on trying to determine the role and importance of extracellular cell wall-degrading enzymes (CWDE) to the virulence of plant pathogenic fungi. Traditionally this has been done by purifying and characterizing CWDE and examining the effect of purified or partially purified enzymes on plant cells. Despite considerable progress, this has not resulted in any definitive conclusions on the importance of CWDE to plant pathogenic fungi. However, in recent years, a new approach using recombinant DNA techniques has been employed to try to provide more conclusive evidence concerning the role of CWDE in plant pathogenesis. This review will examine recent developments in the molecular biology of CWDE of plant pathogenic fungi. Of the numerous CWDE produced by plant pathogenic fungi, most research has concentrated on the pectin degrading enzymes. This is because the pectinases are typically produced first, in the largest amounts, and are the only CWDE capable of macerating plant tissue and killing plant cells on their own (Cooper, 1983). The pectin matrix of plants is found throughout the primary cell wall but is most concentrated in the middle lamella between cells (Carpita and Gibeaut, 1993). The pectin matrix is thought to stabilize cellulose microfibrils, other neutral sugar polymers and proteins in the primary cell wall (Carpita and Gibeaut, 1993). The pectin matrix consists of homogalacturonan and rhamnogalacturonan with various degrees of methyl esterification of the carboxyl group of the galacturonate residues (McNeil et al., 1984). Both polymers have side chains of arabinans, xylans and/or arabinogalactans (McNeil et al., 1984). Fungi produce different types of pectinases that are classified by their substrates, type of lysis and mode of action on the pectin polymer. Unesterified pectate polymers can be degraded by polygalacturonase (PG), which uses hydrolytic cleavage, and pectate lyase (PL), which uses -elimination cleavage and the formation of a double bond in one of the resulting galacturonate residues (Rexová-Benková and Markovič, 1976). Esterified pectin polymers are attacked by pectin lyase (PNL) or polymethylgalacturonase (Rexová-Benková and Markovič, 1976). Rhamnogalacturonase (RHG) cleaves the bond between the alternating galactose and rhamnose residues in rhamnogalacturonan (Suykerbuyk et al., 1995). Two types of pectinases have been differentiated by their cleavage pattern: an endo form

The molecular genetics of virulence of Xanthomonas campestris

Bacteria belonging to the genus Xanthomonas are important pathogens of many plants, and their virulence appears to be due primarily to secreted and surface compounds that could increase host nutrient loss, or avoid or suppress unfavorable conditions in the host. Type II and III secretory pathways are essential for virulence. Some individual extracellular enzymes (type II-secretion dependent) affect final bacterial population levels, whereas some avirulence gene products (type III-secretion dependent) affect virulence by altering host metabolism. Avr proteins, probably secreted via a pilus, can also be recognized by host resistance gene products. Virulence is also associated with bacterial surface polysaccharides, which may help to avoid host defense responses, and regulatory gene systems, which can control virulence gene expression.

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.

Pathotype identification of Leptosphaeria maculans with PCR and oligonucleotide primers from ribosomal internal transcribed spacer sequences

Abstract In order to develop a pathotype-specific assay, the internal transcribed spacer region 1 (ITS 1) of ribosomal RNA genes of highly and weakly virulent isolates of Leptosphaeria maculans were sequenced using primers from the flanking 17S and 5·8S ribosomal DNA (rDNA). The sequenced ITS 1 region had approximately 67% similarity among highly and weakly virulent isolates, and 37–46% similarity between L. maculans and other species of fungi. From the ITS 1 sequences, two pairs of oligonucleotide primers were synthesized which specifically amplified DNA from either the highly or weakly virulent pathotype of L. maculans using the polymerase chain reaction (PCR). Primer pair HV17S/5·8S was based on sequences from the highly virulent isolates, Leroy and LM26, and only amplified highly virulent isolates. Primer pair WV17S/5·8S was based on sequences from the weakly virulent isolate, Unity, and only amplified weakly virulent isolates. This PCR-based assay utilizing pathotype-specific primers was employed to detect an isolate of the highly virulent pathotype of L. maculans in infected plant tissue and shows promise as a diagnostic tool.

Hemibiotrophic infection and identity of the fungus, Colletotrichum destructivum , causing anthracnose of tobacco

The causal agent of tobacco anthracnose was identified as Colletotrichum destructivum based on the morphology of the fungus and a comparison of the sequence of the rDNA ITS with those of other Colletotrichum species. The infection process on tobacco (Nicotiana tabacum and N. benthamiana) was examined by light microscopy, which revealed that the pathogen acted as an intracellular hemibiotroph. Penetration occurred preferentially at the anticlinal walls of epidermal cells by an appressorium and penetration peg. An infection vesicle formed in the penetrated host cell by 48 h after inoculation, and out of this, a multi-lobed infection vesicle grew which remained limited to the initially infected cell. The interaction at this point was biotrophic, which was confirmed by plasmolysis and accumulation of a vital stain by the infected host cells. Thin secondary hyphae arose from multi-lobed infection vesicles at 60 h after inoculation, which then penetrated the host cell wall and began the necrotrophic phase of the infection. Acervuli formed on the plant surface by 96 h after inoculation, typically with a single melanized seta. In addition to tobacco, the fungus could infect alfalfa, cowpea, and Medicago truncatula, but not soybean. The process of infection of C. destructivum in tobacco was very similar to that previously reported in alfalfa and cowpea.

Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal®, ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round-leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72–96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96–120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non-specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.

Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software

A digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. Images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, Scion Image, to measure changes in leaf color caused by fungal sporulation or tissue damage. High correlations were observed between the percent diseased leaf area estimated by Scion Image analysis and the percent diseased leaf area from leaf drawings. These drawings of various foliar diseases came from a disease key previously developed to aid in visual estimation of disease severity. For leaves of Nicotiana benthamiana inoculated with different spore concentrations of the anthracnose fungus Colletotrichum destructivum, a high correlation was found between the percent diseased tissue measured by Scion Image analysis and the number of leaf spots. The method was adapted to quantify percent diseased leaf area ranging from 0 to 90% for anthracnose of lily-of-the-valley, apple scab, powdery mildew of phlox and rust of golden rod. In some cases, the brightness and contrast of the images were adjusted and other modifications were made, but these were standardized for each disease. Detached leaves were used with the flatbed scanner, but a method using attached leaves with a digital camera was also developed to make serial measurements of individual leaves to quantify symptom progression. This was successfully applied to monitor anthracnose on N. benthamiana leaves. Digital image analysis using Scion Image software is a useful tool for quantifying a wide variety of fungal interactions with plant leaves.

A comparison of the pectate lyase genes, pel-1 and pel-2, of Colletotrichum gloeosporioides f.sp. malvae and the relationship between their expression in culture and during necrotrophic infection.

Extracellular pectic lyase and polygalacturonase activities of Colletotrichum gloeosporioides f.sp. malvae were detected in broths containing mallow cell wall extract, pectin or glucose as the carbon source. The initial pH of the broth as well as the carbon source had major influences on pectinase enzyme activities. In the host, only pectic lyase activity was detected, which began at the end of the biotrophic phase and increased in the necrotrophic phase of infection. Two full-length pectate lyase cDNAs, pel-1 and pel-2, were cloned from the fungus. Both genes showed similar patterns of expression when the fungus was grown in mallow cell-wall extract and pectin medium, and the only major difference in expression in culture was that only pel-2 was expressed in glucose broth. Expression of pel-1 and pel-2 was also affected by the initial pH of the medium. Expression of pel-2, but not pel-1, was detected during infection of the host, round-leaved mallow, Malva pusilla. Transcripts of pel-2 were first detectable during the necrotrophic phase of infection approx. 24h after the first detection of pectic lyase enzyme activity. A comparison of expression of pel-1 and pel-2 in culture and in planta with other pectinase genes of C. gloeosporioides f.sp. malvae, as well as with other plant pathogenic fungi, indicates that expression during necrotrophic infection correlates with the ability to be expressed in media containing glucose.

Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae.

Abstract. Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72–96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction.

Hemibiotrophic infection of round-leaved mallow by Colletotrichum gloeosporioides f. sp. malvae in relation to leaf senescence and reducing reagents

The infection of round-leaved mallow ( Malva pusilla ) leaves by Colletotrichum gloeosporioides f. sp. malvae was studied using light and confocal microscopy. Conidia germinated and produced appressoria within 24 h after inoculation. An infection peg arose from the base of the appressorium and directly penetrated an epidermal cell. An intracellular infection vesicle appeared beneath the penetration site by 48 h after inoculation. Large primary hyphae (LPH, approx. 4 μ diam.) emerged from the vesicle and grew intracellularly through several adjacent epidermal cells and then intercellularly between mesophyll cells. Epidermal cells infected by LPH maintained their viability, as shown by their ability to plasmolyse and accumulate neutral red stain. No visible disease symptoms appeared during this biotrophic stage of infection. Thin secondary hyphae (TSH, approx. 2 μ diam.) developed from LPH in 4–5 days after inoculation and were associated with the appearance of necrotic lesions. Host cell wall maceration was visible only during the necrotrophic stage. The duration of the biotrophic stage decreased as mallow leaves became older or were senesced by placing them in the dark. TSH and host cell necrosis developed as soon as 48 h after inoculation of the most senescent leaves, and TSH were observed directly emerging from the infection vesicle. Application of thiol reagents, such as glutathione and dithiothreitol, lengthened the biotrophic stage and delayed symptom development, whereas an antioxidant, ascorbate, enhanced infection and promoted symptom development.