Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.     

Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue

A cDNA clone (TAB7) encoding a putative woundinduced (Win) proteinhas been isolated from a tomato (Lycopersicon esculentum Mill.cv. Ailsa Craig) leaf abscission zone cDNA library using a differentialscreening strategy. The clone has a high degree of homologyat the amino acid level to both the potato win1 and 2 genes,Hevea brasiliensis hevein and Nicotiana tabacum PR-4a and PR-4bproteins. The mRNA encoded by TAB7 is up-regulated within 12h of exposure to ethylene (10µl l^–1) and its expressionincreases steadily within the cells comprising the leaf abscissionzone and to a lesser extent in the adjacent non-zone tissue.This rise precedes the onset of cell separation. Southern analysisindicates that the mRNA is encoded by either a single gene ora small gene family. The role of the protein during abscissionis discussed. Key words: Lycopersicon esculentum, abscission zone, ethylene, tomato, wound-induced proteins     

Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2

Summary The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequencing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 protein sequence was found to be related to a family of proteins including hevein and Win-1, which have an amino-terminal lectin domain and a carboxy-terminal domain of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses characterized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dramatically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Pathogenesis-related proteins in lima bean leaves infected with tobacco ringspot virus and their distribution within and around local lesions

Tobacco ringspot virus (TRSV) induces circular, darkbrown local lesions on primary leaves of lima bean (Phaseolus lunatus cv Nemagreen) with a concomitant production of three basic and three acidic pathogenesisrelated (PR) proteins. The three basic proteins are: a 21 kDa protein related serologically to Pinto bean PR-4d and tobacco PR-5 proteins; a 36 kDa glucanase that is related to tobacco PR-2; and, a 31 kDa chitinase related serologically to ethylene-induced bean chitinase. The three acidic 18 kDa lima bean PR proteins are serologically similar and probably are charged isomers of the same protein. The 21 kDa basic protein and the 18 kDa acidic protein accumulated preferentially at the lesion center while the 31 kDa chitinase and TRSV were distributed evenly throughout the necrotic area. In green tissue immediately surrounding a lesion, the amounts of PR proteins were comparable to or lower than those in the necrotic area, and virions were not detected. This mode of spatial distribution indicates that lima bean PR proteins are not involved in TRSV localization, and is consistent with other observations that PR proteins play no direct role in restricting viral spread.     

Organization of phenylalanine ammonia lyase (<Emphasis Type="Italic">PAL</Emphasis>), acidic <Emphasis Type="Italic">PR-5</Emphasis> and osmotin-like (<Emphasis Type="Italic">OSM</Emphasis>) defence-response gene families in the potato genome

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing ~50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.     

Expression of XET-related genes and its relation to elongation in leaves of barley (Hordeum vulgare L.)

Five cDNA clones were isolated from barley (Hordeum vulgare L.) that encoded mRNAs related to xyloglucan endotransglycosylase (XET). One of the clones encoded a protein with XET activity in vitro. Sequence comparisons revealed five families of XET-related sequences, one of which (containing two of the barley genes) was novel. Hybridization studies using clone-specific probes indicated that the corresponding genes were represented once, or possibly twice, in the barley genome. Treatment of dwarf mutants with gibberellic acid (GA₃), or homozygosity at the ‘slender’ (sln1) locus, resulted in a 2.5-fold (approximately) stimulation of blade elongation rate. Three of the five clones detected mRNAs that were maximally expressed towards the base of the blade, and present in greater quantities in GA₃-treated or slender seedlings. The remaining two clones detected mRNAs that were maximally expressed in the middle of the blade. Relative elemental growth rate (REGR) profiles of leaves growing with or without GA₃ treatment revealed similar maximal REGR values despite a 2.5-fold difference in leaf elongation rate. Segments of GA₃-treated leaves attained their maximal REGR values more rapidly, this being associated with enhanced expression of the three ‘basal’ XET-related mRNAs. Highest XET activities were detected in the base of the elongation zone, and in GA₃-treated seedlings a second activity peak was observed near the distal end of the elongation zone. We conclude that there are likely to be several XET isoenzymes with different expression patterns, and identify those XET-related proteins potentially involved in leaf elongation.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed. Received: 25 May 1998 / Accepted: 29 June 1998     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf.

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR-4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI approximately 9.3, M(r) approximately 13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using approximately 10 micrograms/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR-3 type protein) or with barley protein R (a PR-5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.     

Isolation and characterization of cDNA clones corresponding to the genes expressed preferentially in floral organs of Arabidopsis thaliana

Seventeen cDNA clones of genes corresponding to mRNAs expressed preferentially in floral organs of Arabidopsis thaliana were obtained by differential screening of a flower bud cDNA library, and classified into five groups (1A, 17A, 1B, 4B and 5B) by cross-hybridization and restriction analysis. Sequence analysis revealed that the 1A-1 and 17A-1 clones encode vegetative storage proteins (VSPs). The VSP mRNAs were detected in a small amount in leaves and increased to a limited level by wounding. Both 1B-1 and 5B-1 clones were homologous to transmembrane protein cDNAs. The protein encoded by 4B-1 clone contained a proline-rich region, but no homologous proteins were found in databases.     

cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products

A cDNA library was constructed to 10-15 S poly(A) RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco. By differential colony hybridization of 1400 transformants, 32 clones were obtained corresponding to TMV-inducible tobacco mRNAs. These clones were subdivided into six clusters on the basis of cross-hybridization of the inserts. By Northern blot hybridization it was shown that three of the corresponding mRNAs were strongly induced by spraying tobacco plants with salicylic acid, whereas one mRNA was weakly induced by this treatment. All mRNAs were systemically induced in plants in which only the lower leaves were locally infected by TMV. Hybrid-selected translation was performed, using six clones representing one cluster each, followed by immunoprecipitation using an antiserum to purified pathogenesis-related (PR) proteins. Four clones yielded precipitable translation products. One of these clones represented a cluster of PR-1 clones, another clone encoded the thaumatin-like (TL) protein of tobacco which may correspond to PR-P or −Q.     

Translatable mRNA changes in ethylene induced abscission zones of Phaseolus vulgaris (Red Kidney)

Abstract The wheat germ translation system was programmed with soluble RNA extracted from foliar abscission zones of Phaseolus vulgaris, These extracts were taken at various times after the induction of abscission. A translation product with a molecular weight of 42 kilodalton (kD) was only present after this treatment, though three other species 32, 27 and 17 kD increased substantially. The isozyme of cellulase with a pi of 9.5 could not be conclusively identified amongst the products though the 32 kD protein is probably chitinase. Comparison of the abscission zone translatable RNA with that from adjacent petiole and stem tissues showed the 17 kD protein developed in all these location. The 42, 32 and 27 kD bands were found predominantly in the zone and petiole.     

Cytokinin stress changes the developmental regulation of several defence-related genes in tobacco

Tobacco shoots exposed to elevated endogenous or exogenous cytokinin levels are unable to develop roots and lack apical dominance. We have isolated cDNA copies of five mRNA species that accumulate to elevated levels in such cytokinin-stressed shoots via differential screening of a cDNA library of transgenic shoots which contain an active T-DNA cytokinin gene (T-cyt gene) from Agrobacterium tumefaciens. Four of the cDNA clones were found to correspond to plant defence-related mRNAs, encoding extensin, chitinase, PR-1 and a PR-1-like protein, respectively. In normal tobacco plants PR-1 mRNA is relatively rare in all organs. The other four mRNAs occur at relatively low levels in shoots, especially in leaves, but are very prevalent in roots. Extensin mRNA, for example, is not detectable in leaves, while it is an abundant mRNA in roots and stems. In normal shoots cultured on cytokinin-containing medium all five mRNAs accumulate to elevated levels, similar to those found in transgenic T-cyt shoots. We conclude that the imposed cytokinin stress causes changes in the tissue-specific control of the levels of several defence-related mRNA species in tobacco.     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.