Artur R. Teixeira

The role of plant defence proteins in fungal pathogenesis.

SUMMARY It is becoming increasingly evident that a plant-pathogen interaction may be compared to an open warfare, whose major weapons are proteins synthesized by both organisms. These weapons were gradually developed in what must have been a multimillion-year evolutionary game of ping-pong. The outcome of each battle results in the establishment of resistance or pathogenesis. The plethora of resistance mechanisms exhibited by plants may be grouped into constitutive and inducible, and range from morphological to structural and chemical defences. Most of these mechanisms are defensive, exhibiting a passive role, but some are highly active against pathogens, using as major targets the fungal cell wall, the plasma membrane or intracellular targets. A considerable overlap exists between pathogenesis-related (PR) proteins and antifungal proteins. However, many of the now considered 17 families of PR proteins do not present any known role as antipathogen activity, whereas among the 13 classes of antifungal proteins, most are not PR proteins. Discovery of novel antifungal proteins and peptides continues at a rapid pace. In their long coevolution with plants, phytopathogens have evolved ways to avoid or circumvent the plant defence weaponry. These include protection of fungal structures from plant defence reactions, inhibition of elicitor-induced plant defence responses and suppression of plant defences. A detailed understanding of the molecular events that take place during a plant-pathogen interaction is an essential goal for disease control in the future.

The wine proteins

Proteins are typically present in wines in low concentrations, contributing little to their nutritive value. However, they assume a considerable technological and economical importance because they greatly affect the clarity and stability of wines. Although exhibiting a large diversity, the majority of the wine proteins are structurally related and have been identified as pathogenesis related (PR) proteins. Thus, different wines are essentially composed by identical sets of polypeptides. They derive from the grape pulp, and survive the vinification process simply because they are highly resistant to proteolysis and to the low pH characteristic of wines. There is increasing evidence suggesting that although protein-dependent, the development of turbidity in wines is controlled by a number of factors of non-protein origin, such as polyphenols, the wine pH and the presence of polysaccharides. A variety of procedures has been developed and tested for the specific removal of proteins from wines. Even though bentonite fining is nonspecific and can impair the quality of wine, it remains the only effective method to stabilize wines.

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong−type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.

Use of a single method in the extraction of the seed storage globulins from several legume species. Application to analyse structural comparisons within the major classes of globulins.

In this study, a single, improved methodology was used to extract, fractionate and purify the 11S (legumin-type or related to the alpha-conglutin from Lupinus albus L.), 7S (vicilin-type or related to the beta-conglutin from L. albus) and 2S (related to the gamma-conglutin from L. albus) families of proteins from eight legume species: L. albus, Glycine max (L.) Merr., Pisum sativum L., Vicia faba L., Cicer arietinum L., Phaseolus vulgaris L., Lens culinaris Med. and Arachis hypogaea L. The sedimentation coefficients obtained varied from 1.9 to 8.1 for the gamma-conglutin-related proteins, from 5.1 to 10.5 for the beta-conglutin-related proteins and from 12.0 to 14.9 for the alpha-conglutin-related globulins. The gamma-conglutin-related proteins is the most heterogeneous group. Antibodies produced against each type of gamma-conglutin polypeptide chain recognize the other polypeptide chain as well as other polypeptides in the corresponding globulins from all species examined. The 7S globulins are typically composed of a large number of polypeptides, covering a wide range of molecular masses (10 to 70 kD). The presence of disulphide bonds is apparently absent and the occurrence of glycopolypeptides is not widespread. Finally, the 11S globulins are characteristically formed by a limited number of polypeptides that may be divided into a lighter group (20-25 kD) and a heavier group (35-50 kD). The presence of disulphide bonds is apparently widespread but the occurrence of glycopolypeptides seems to be relatively rare. Both the 7S family and the 11S globulins studied by immunoblotting exhibit a low level of structural similarity.In this study, a single, improved methodology was used to extract, fractionate and purify the 11S (legumin-type or related to the α-conglutin from Lupinus albus L.), 7S (vicilin-type or related to the β-conglutin from L. albus) and 2S (related to the γ-conglutin from L. albus) families of proteins from eight legume species: L. albus, Glycine max (L.) Merr., Pisum sativum L., Vicia faba L., Cicer arietinum L., Phaseolus vulgaris L., Lens culinaris Med. and Arachis hypogaea L. The sedimentation coefficients obtained varied from 1.9 to 8.1 for the γ-conglutinrelated proteins, from 5.1 to 10.5 for the β-conglutin-related proteins and from 12.0 to 14.9 for the α-conglutin-related globulins. The γ-conglutin-related proteins is the most heterogenous group. Antibodies produced against each type of γ-conglutin polypeptide chain recognize the other polypeptide chain as well as other polypeptides in the corresponding globulins from all species examined. The 7S globulins are typically composed of a large number of polypeptides, covering a wide range of molecular masses (10 to 70 kD). The presence of disulphide bonds is apparently absent and the occurrence of glycopolypeptides is not widespread. Finally, the 11S globulins are characteristically formed by a limited number of polypeptides that may be divided into a lighter group (20-25 kD) and a heavier group (35-50 kD). The presence of disulphide bonds is apparently widespread but the occurrence of glycopolypeptides seems to be relatively rare. Both the 7S family and the 11S globulins studied by immunoblotting exhibit a low level of structural similarity.

Lupinus luteus, Vicia sativa and Lathyrus cicera as protein sources for piglets: ileal and total tract apparent digestibility of amino acids and antigenic effects

Abstract Twenty-four male piglets, weaned at 28 days of age, were used to measure the total and ileal digestibility and serum immune responses to dietary leguminous seeds. The experimental diets consisted of a control starter (C) and three other diets prepared by replacing 30% of the crude protein content of the C diet by the protein of Lupinus luteus (LL), Vicia sativa (VS) or Lathyrus cicera (LC). The total tract apparent digestibility (TTAD) of energy and crude protein (CP) was lowest ( P P L. luteus , vicilin of V. sativa and vicilin of L. cicera were detected 28 days after feeding the diet in the sera of piglets fed on the LL, VS or LC diets, respectively. Conversely, no storage protein was found in the serum of any piglet fed either on LL, VS or LC diets. The presence of antibodies against β-conglutin of lupine, vicilin of V. sativa and vicilin of L. cicera , respectively indicated an immune response in weaned piglets. The absence of residual antigenic proteins may be due to the digestive adaptation of the piglet to the legume-based diets. However, no direct relation between the differences in digestibility coefficients among the legume seeds and their antigenicity was established.

Accumulation of a lectin-like breakdown product of β-conglutin catabolism in cotyledons of germinating Lupinus albus L. seeds

Abstract. During germination of Lupinus albus seeds, a 20-kDa polypeptide accumulates in the cotyledons of 4-d-old plants (Ferreira et al., 1995b, J Exp Bot 46: 211–219). Immunological, polypeptide cleavage with cyanogen bromide and amino acid sequencing experiments indicate that the 20-kDa polypeptide and ubiquitin are structurally unrelated. However there is a strong sequence homology between the 20-kDa polypeptide and the vicilin-like storage proteins from pea and soybean. Our results indicate that the 20-kDa polypeptide is an intermediate breakdown product of β-conglutin catabolism, the vicilin-like storage protein from L. albus, and that its interaction with anti-ubiquitin antibodies results from the recognition of the antibodies by the 20-kDa polypeptide rather than by the opposite. Besides rabbit anti-ubiquitin antibodies, the 20-kDa polypeptide interacts with a variety of glycoproteins, including immunoglobulin G from several animal species, peroxidase and alkaline phosphatase, suggesting that it possesses a lectin-type activity. Its activity is resistant to sodium dodecyl sulfate or methanol treatments, boiling and autoclaving. Purification of the 20-kDa polypeptide and immunological studies with anti-20-kDa-polypeptide antibodies showed that the non-glycosylated polypeptide is part of a glycoprotein with an estimated molecular mass of 210 kDa, composed of several types of structurally related subunit with molecular masses ranging from 14 to 50 kDa. Purified native protein containing the 20-kDa polypeptide self-aggregates in a calcium-dependent manner as reported for some glycosylated lectins. The possible physiological function of the 20-kDa polypeptide is discussed.

Self‐aggregation of legume seed storage proteins inside the protein storage vacuoles is electrostatic in nature, rather than lectin‐mediated

Conglutins are multisubunit, glycosylated, major storage proteins present in Lupinus seeds that self‐aggregate in a calcium/magnesium‐dependent manner. Two of these globulins exhibit lectin activity. The 210 kDa globulin derived from β‐conglutin that accumulates in Lupinus cotyledons during germination was used as a model protein to establish whether the self‐aggregation process is electrostatic in nature or lectin‐mediated. This protein binds in a very strong manner to chitin and recognizes a variety of glycoproteins including immunoglobulins G. Several compounds were tested for their inhibitory effect on the cation‐dependent self‐aggregation process. Sialic acid and phytin were the most effective whereas chitin and mucin were totally ineffective. The inability of the oligosaccharidic side chains of the 210 kDa protein, β‐conglutin and immunoglobulin G to interfere with the aggregation strongly supports the view that Ca/Mg are electrostatically involved in the in vitro self‐aggregation of Lupinus globulins. The results suggest that calcium and magnesium ions are also electrostatically involved in vivo in the macromolecular aggregation of legume seed storage proteins, ensuring their efficient packing inside the protein storage vacuoles. This mechanism is responsible for the typical insolubility of legume globulins in water.

Protein degradation in C3 and C4 plants subjected to nutrient starvation. Particular reference to ribulose bisphosphate carboxylase/oxygenase and glycolate oxidase

Abstract In a previous study [Esquivel et al., J. Exp. Bot. 49 (1998) 807–816], the degradation patterns of ribulose bisphosphate carboxylase/oxygenase (RuBP carboxylase/oxygenase; EC4.1.1.39) and of glycolate oxidase (EC1.1.3.1) were shown to be species specific under normal metabolic conditions, suggesting that they do not depend on the type of photosynthetic metabolism. In this work, we have extended this study to analyse protein degradation under conditions of sulphur or nitrogen deprivation in the second leaves of intact wheat (C 3 ), maize (C 4 ) and sorghum (C 4 ) plants. The plants were grown and the leaf proteins double-labelled with radioactive precursors. Immediately after the labelling period, the plants were transferred to unlabelled medium and chased, under selected conditions, for up to 15 days to measure protein turnover. Sulphur and nitrogen deficiencies greatly reduce plant growth. However, the fresh weight of the second leaves utilised in the experiments remain essentially unaltered during the 15-day chase period. Changes in chlorophyll, total soluble protein and RuBP carboxylase/oxygenase activity in the plants grown under control conditions, or deprived of sulphur or nitrogen, produce similar patterns in the three species examined. Under sulphur deficiency, degradation of wheat RuBP carboxylase/oxygenase is faster than that of the total soluble protein. However, sulphur deficiency in the two C 4 plants does not affect the rate of enzyme catabolism. On the other hand, nitrogen deprivation slightly increases the degradation of wheat and sorghum RuBP carboxylase/oxygenases, but strongly enhances the degradation of the enzyme in maize. Glycolate oxidase appears to be a fast turning over enzyme in all plants tested, exhibiting a large enhancement in its rate of degradation with sulphur or nitrogen deficiency. The results obtained suggest that in C 3 plants, total soluble protein, RuBP carboxylase/oxygenase and glycolate oxidase degradation are more affected by sulphur deficiency than by nitrogen deprivation. The opposite appears to be true for C 4 plants. These observations raise the possibility that these responses may be somewhat dependent on the type of photosynthetic metabolism, but raise uncertainties about considering RuBP carboxylase/oxygenase as a leaf storage protein.

Legume Proteins of the Vicilin Family are More Immunogenic Than Those of the Legumin Family in Weaned Piglets

Two experiments were conducted to study the systemic and local immune responses to legume proteins in piglets weaned at 28 d of age. The pigs were fed a control diet (C) based on casein or diets in which 50% of crude protein (CP) was supplied by soya bean meal (SBM), pea (P), faba bean (FB), or lupin (L) seeds in experiment 1, and white chickpea (WCP) or black chickpea (BCP) seeds in experiment 2. Blood samples and intestinal secretions were collected at slaughter four weeks later and analysed for specific antibodies using ELISA and western blotting. Piglets fed the legume-containing diets had higher ( P < 0.10) plasma immunoglobulin (Ig) G ELISA titres to legume proteins than did piglets fed a casein-based diet. Many protein bands in the molecular weight range of 94.0-14.4 kDa were recognized by plasma IgG and IgA antibodies. The most immunogenic bands belonged to proteins of the vicilin family and to other unidentified minor proteins. No specific IgA antibodies were detected in intestinal secretions by ELISA and western blotting.

The unique biosynthetic route from Lupinus β-conglutin gene to blad.

Background During seed germination, β-conglutin undergoes a major cycle of limited proteolysis in which many of its constituent subunits are processed into a 20 kDa polypeptide termed blad. Blad is the main component of a glycooligomer, accumulating exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Principal Findings The sequence of the gene encoding β-conglutin precursor (1791 nucleotides) is reported. This gene, which shares 44 to 57% similarity and 20 to 37% identity with other vicilin-like protein genes, includes several features in common with these globulins, but also specific hallmarks. Most notable is the presence of an ubiquitin interacting motif (UIM), which possibly links the unique catabolic route of β-conglutin to the ubiquitin/proteasome proteolytic pathway. Significance Blad forms through a unique route from and is a stable intermediary product of its precursor, β-conglutin, the major Lupinus seed storage protein. It is composed of 173 amino acid residues, is encoded by an intron-containing, internal fragment of the gene that codes for β-conglutin precursor (nucleotides 394 to 913) and exhibits an isoelectric point of 9.6 and a molecular mass of 20,404.85 Da. Consistent with its role as a storage protein, blad contains an extremely high proportion of the nitrogen-rich amino acids.