Antimicrobial Phytoprotectants and Fungal Pathogens: A Commentary

Many plants produce antifungal secondary metabolites. These may be preformed compounds which are found in healthy plants and which may represent in-built chemical barriers to infection by potential pathogens (preformed antimicrobial compounds or phytoanticipins). Alternatively they may be synthesized in response to pathogen attack as part of the plant defence response (phytoalexins). If these molecules do play a role in protecting plants against pathogen attack, then successful pathogens are presumably able to circumvent or tolerate these defences. Strategies may include avoidance, enzymatic degradation, and/or nondegradative mechanisms. This review outlines the different ways in which fungal pathogens may counter the antifungal compounds produced by their host plants and summarizes the evidence for and against these compounds as antimicrobial phytoprotectants.     

Mutualism versus pathogenesis: the give-and-take in plant–bacteria interactions

Pathogenic bacteria and mutualistic rhizobia are able to invade and establish chronic infections within their host plants. The success of these plant–bacteria interactions requires evasion of the plant innate immunity by either avoiding recognition or by suppressing host defences. The primary plant innate immunity is triggered upon recognition of common microbe-associated molecular patterns. Different studies reveal striking similarities between the molecular bases underlying the perception of rhizobial nodulation factors and microbe-associated molecular patterns from plant pathogens. However, in contrast to general elicitors, nodulation factors can control plant defences when recognized by their cognate legumes. Nevertheless, in response to rhizobial infection, legumes show transient or local defence-like responses suggesting that Rhizobium is perceived as an intruder although the plant immunity is controlled. Whether these responses are involved in limiting the number of infections or whether they are required for the progression of the interaction is not yet clear. Further similarities in both plant–pathogen and Rhizobium –legume associations are factors such as surface polysaccharides, quorum sensing signals and secreted proteins, which play important roles in modulating plant defence responses and determining the outcome of the interactions.     

Modular structure of HEL protein from Arabidopsis reveals new potential functions for PR-4 proteins

Abstract Plants possess an innate immune system enabling them to defend themselves against pathogen attack. The accumulation of newly synthesized pathogenesis-related proteins (PRs) is one of the most studied inducible plant defence response. In this paper, we report on the characterization of a class I PR4 vacuolar protein from Arabidopsis, named AtHEL. The protein has a modular structure consisting of an N-terminal hevein-like domain (CB-HEL) and a C-terminal domain (CD-HEL) that are posttranslationally processed. Both domains show a strong antifungal activity, but they do not have chitinolitic properties. CD-HEL was found to be endowed with RNase, but not DNase activity. Molecular modeling carried out on both domains revealed that CB-HEL possesses a chitin binding site strictly conserved between hevein-type peptides and that the cavity involved in substrate interaction of CD-HEL do not show any residue substitution with respect to the orthologous wheatwin1 from wheat. Using a fishing for partners approach, CB-HEL was found to interact with a fungal fruiting body lectin. According to literature, we can hypothesize that CB-HEL could cross the pathogen hyphal membrane and that its interaction with a fungal lectin could knock out one of the weapons that the fungus uses.     

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.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) expressing tomato glucanase leads to arrested growth of <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.     

The plant hormone salicylic acid interacts with the mechanism of anti‐herbivory conferred by fungal endophytes in grasses

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal‐produced alkaloids. Because of the role that alkaloids play in anti‐herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N‐formyllolines and N‐acetylnorlolines) and consequently decreased the endophyte‐conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte‐conferred resistance against herbivores.     

Salicylic acid: an old hormone up to new tricks

Salicylic acid (SA) acts as a signalling molecule in plant defence against biotrophic and hemibiotrophic phytopathogens. The biosynthesis of SA on pathogen detection is essential for local and systemic acquired resistance, as well as the accumulation of pathogenesis‐related (PR) proteins. SA biosynthesis can occur via several different substrates, but is predominantly accomplished by isochorismate synthase (ICS1) following pathogen recognition. The roles of BTB domain‐containing proteins, NPR1, NPR3 and NPR4, in SA binding and signal transduction have been re‐examined recently and are elaborated upon in this review. The pathogen‐mediated manipulation of SA‐dependent defences, as well as the crosstalk between the SA signalling pathway, other plant hormones and defence signals, is also discussed in consideration of recent research. Furthermore, the recent links established between SA, pathogen‐triggered endoplasmic reticulum stress and the unfolded protein response are highlighted.     

Heterologous chitinase gene expression to improve plant defense against phytopathogenic fungi

Agricultural crops worldwide suffer from a vast array of fungal diseases which cause severe yield losses. Upon interaction with a pathogen, plants initiate a complex network of defense mechanisms, among which is a dramatic increase in chitinase activity. Chitinases are capable of hydrolyzing chitin-containing fungal cell walls and are therefore thought to play a major role in the plant’s response. One of the strategies to increase plant tolerance to fungal pathogens is the constitutive overexpression of proteins involved in plant-defense mechanisms. The level of protection observed in transgenic plants harboring heterologous chitinase genes varies, depending on the particular combination of enzyme, plant and pathogen tested. Nevertheless, most of these transgenic plants exhibit increased tolerance to fungal diseases relative to their non-transgenic counterparts. The combined expression of chitinases with other plant-defense proteins such as glucanases and ribosome-inactivating proteins further enhances the plant’s resistance to fungal attack. Received 29 January 1997/ Accepted in revised form 01 July 1997     

Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis

The deposition of lignin during plant–pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D , act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato , possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.     

The effects of extracellular adenosine 5′‐triphosphate on the tobacco proteome

Extracellular adenosine 5′‐triphosphate (eATP) is emerging as an important plant signalling compound capable of mobilising intracellular second messengers such as Ca²⁺, nitric oxide, and reactive oxygen species. However, the downstream molecular targets and the spectrum of physiological processes that eATP regulates are largely unknown. We used exogenous ATP and a non‐hydrolysable analogue as probes to identify the molecular and physiological effects of eATP‐mediated signalling in tobacco. 2‐DE coupled with MS/MS analysis revealed differential protein expression in response to perturbation of eATP signalling. These proteins are in several functional classes that included photosynthesis, mitochondrial ATP synthesis, and defence against oxidative stress, but the biggest response was in the pathogen defence‐related proteins. Consistent with this, impairment of eATP signalling induced resistance against the bacterial pathogen Erwinia carotovora subsp. carotovora. In addition, disease resistance activated by a fungal pathogen elicitor (xylanase from Trichoderma viride) was concomitant with eATP depletion. These results reveal several previously unknown putative molecular targets of eATP signalling, which pinpoint eATP as an important hub at which regulatory signals of some major primary metabolic pathways and defence responses are integrated.     

Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.     

Treating seeds with activators of plant defence generates long-lasting priming of resistance to pests and pathogens

? Priming of defence is a strategy employed by plants exposed to stress to enhance resistance against future stress episodes with minimal associated costs on growth. Here, we test the hypothesis that application of priming agents to seeds can result in plants with primed defences. ? We measured resistance to arthropod herbivores and disease in tomato (Solanum lycopersicum) plants grown from seed treated with jasmonic acid (JA) and/or β-aminobutryric acid (BABA). ? Plants grown from JA-treated seed showed increased resistance against herbivory by spider mites, caterpillars and aphids, and against the necrotrophic fungal pathogen, Botrytis cinerea. BABA seed treatment provided primed defence against powdery mildew disease caused by the biotrophic fungal pathogen, Oidium neolycopersici. Priming responses were long-lasting, with significant increases in resistance sustained in plants grown from treated seed for at least 8 wk, and were associated with enhanced defence gene expression during pathogen attack. There was no significant antagonism between different forms of defence in plants grown from seeds treated with a combination of JA and BABA. ? Long-term defence priming by seed treatments was not accompanied by reductions in growth, and may therefore be suitable for commercial exploitation.     

Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora

Plant calcium can modulate a particular plant–pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora , the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora , the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant–pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.     

The ubiquitin/26S proteasome system in plant–pathogen interactions: a never-ending hide-and-seek game

The ubiquitin/26S proteasome system (UPS) plays a central role in plant protein degradation. Over the past few years, the importance of this pathway in plant–pathogen interactions has been increasingly highlighted. UPS is involved in almost every step of the defence mechanisms in plants, regardless of the type of pathogen. In addition to its proteolytic activities, UPS, through its 20S RNase activity, may be part of a still unknown antiviral defence pathway. Strikingly, UPS is not only a weapon used by plants to defend themselves, but also a target for some pathogens that have evolved mechanisms to inhibit and/or use this system for their own purposes. This article attempts to summarize the current knowledge on UPS involvement in plant–microbe interactions, a complex scheme that illustrates the never-ending arms race between hosts and microbes.     

Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

1.  Plants are simultaneously attacked by multiple herbivores and pathogens. While some plant defences act synergistically, others trade-off against each other. Such trade-offs among resistances to herbivores and pathogens are usually explained by the costs of resistance, i.e. resource limitations compromising a plant's overall defence.
2.  Here, we demonstrate that trade-offs can also result from direct negative interactions among defensive traits. We studied cyanogenesis (release of HCN) of lima bean (Fabaceae: Phaseolus lunatus ) and effects of this efficient anti-herbivore defence on resistance to a fungal pathogen (Melanconiaceae: Colletotrichum gloeosporioides ).
3.  Leaf tissue destruction by fungal growth was significantly higher on high cyanogenic (HC) lima bean accessions than on low cyanogenic (LC) plants. The susceptibility of HC accessions to the fungal pathogen was strongly correlated to reduced activity of resistance-associated polyphenol oxidases (PPOs) in leaves of these plants. LC accessions, in contrast, showed high PPO activity, which was correlated with distinct resistance to C. gloeosporioides .
4.  Experimentally applied, gaseous HCN reduced PPO activity and significantly increased the size of lesions caused by C. gloeosporioides in LC leaves.
5.  Field observations of a wild lima bean population in Mexico revealed a higher infection rate of HC compared to LC plant individuals. The types of lesions observed on the different cyanogenic plants in nature were similar to those observed on HC and LC plants in the laboratory.
6.   Synthesis. We suggest that cyanogenesis of lima bean directly trades off with plant defence against fungal pathogens and that the causal mechanism is the inhibition of PPOs by HCN. Our findings provide a functional explanation for the observed phenomenon of the low resistance of HC lima beans in nature.     

Fungal Resistance to Plant Antibiotics as a Mechanism of Pathogenesis

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.     

Fungal resistance to plant antibiotics as a mechanism of pathogenesis.

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.     

Structural basis of the antifungal activity of wheat PR4 proteins

PR4 proteins possess antifungal activity against several pathogenic fungi suggesting a pivotal role in defence reactions against plant pathogen attack. We already showed that wheatwin1, a wheat PR protein of class 4, is endowed with ribonuclease activity. In this study we produced three mutants altering the active site and performed comparative analysis with the native protein also in the presence of the ribonuclease inhibitor 5′-ADP. We characterized the RNA binding site and its interaction with 5′-ADP by 3D modelling and docking studies. Moreover, in vitro antifungal assays have been carried out in order to study the relationship between antifungal and ribonuclease activities. Finally, localization of wheatwin1 in Fusarium culmorum spores was evaluated using fluorescence light microscope.     

Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi

β-1,3-Glucanases are abundant in plants and have been characterized from a wide range of species. They play key roles in cell division, trafficking of materials through plasmodesmata, in withstanding abiotic stresses and are involved in flower formation through to seed maturation. They also defend plants against fungal pathogens either alone or in association with chitinases and other antifungal proteins. They are grouped in the PR-2 family of pathogenesis-related (PR) proteins. Use of β-1,3-glucanase genes as transgenes in combination with other antifungal genes is a plausible strategy to develop durable resistance in crop plants against fungal pathogens. These genes, sourced from alfalfa, barley, soybean, tobacco, and wheat have been co-expressed along with other antifungal proteins, such as chitinases, peroxidases, thaumatin-like proteins and α-1-purothionin, in various crop plants with promising results that are discussed in this review.