Carl H. Mesarich

Understanding Plant Immunity as a Surveillance System to Detect Invasion

Various conceptual models to describe the plant immune system have been presented. The most recent paradigm to gain wide acceptance in the field is often referred to as the zigzag model, which reconciles the previously formulated gene-for-gene hypothesis with the recognition of general elicitors in a single model. This review focuses on the limitations of the current paradigm of molecular plant-microbe interactions and how it too narrowly defines the plant immune system. As such, we discuss an alternative view of plant innate immunity as a system that evolves to detect invasion. This view accommodates the range from mutualistic to parasitic symbioses that plants form with diverse organisms, as well as the spectrum of ligands that the plant immune system perceives. Finally, how this view can contribute to the current practice of resistance breeding is discussed.

Venturia inaequalis: the causal agent of apple scab

UNLABELLED The fungus Venturia inaequalis infects members of the Maloideae, and causes the disease apple scab, the most important disease of apple worldwide. The early elucidation of the gene-for-gene relationship between V. inaequalis and its host Malus has intrigued plant pathologists ever since, with the identification of 17 resistance (R)-avirulence (Avr) gene pairings. The Avr gene products are presumably a subset of the total effector arsenal of V. inaequalis (predominantly proteins secreted in planta assumed to facilitate infection). The supposition that effectors from V. inaequalis act as suppressors of plant defence is supported by the ability of the pathogen to penetrate the cuticle and differentiate into large pseudoparenchymatous structures, termed stromata, in the subcuticular space, without the initiation of an effective plant defence response. If effectors can be identified that are essential for pathogenicity, the corresponding R genes will be durable and would add significant value to breeding programmes. An R gene cluster in Malus has been cloned, but no V. inaequalis effectors have been characterized at the molecular level. However, the identification of effectors is likely to be facilitated by the resolution of the whole genome sequence of V. inaequalis. TAXONOMY Teleomorph: Venturia inaequalis Cooke (Wint.); Kingdom Fungi; Phylum Ascomycota; Subphylum Euascomycota; Class Dothideomycetes; Family Venturiaceae; genus Venturia; species inaequalis. Anamorph: Fusicladium pomi (Fr.) Lind or Spilocaea pomi (Fr.). LIFE CYCLE: V. inaequalis is a hemibiotroph and overwinters as pseudothecia (sexual fruiting bodies) following a phase of saprobic growth in fallen leaf tissues. The primary inoculum consists of ascospores, which germinate and penetrate the cuticle. Stromata are formed above the epidermal cells but do not penetrate them. Cell wall-degrading enzymes are only produced late in the infection cycle, raising the as yet unanswered question as to how V. inaequalis gains nutrients from the host. Conidia (secondary inoculum) arise from the upper surface of the stromata, and are produced throughout the growing season, initiating multiple rounds of infection. VENTURIA INAEQUALIS AS A MODEL PATHOGEN OF A WOODY HOST: V. inaequalis can be cultured and is amenable to crossing in vitro, enabling map-based cloning strategies. It can be transformed readily, and functional analyses can be conducted by gene silencing. Expressed sequence tag collections are available to aid in gene identification. These will be complemented by the whole genome sequence, which, in turn, will contribute to the comparative analysis of different races of V. inaequalis and plant pathogens within the Dothideomycetes.

The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.

Candidate effector gene identification in the ascomycete fungal phytopathogen Venturia inaequalis by expressed sequence tag analysis.

The hemi-biotrophic fungus Venturia inaequalis infects members of the Maloideae, causing the economically important apple disease, scab. The plant-pathogen interaction of Malus and V. inaequalis follows the gene-for-gene model. cDNA libraries were constructed, and bioinformatic analysis of the resulting expressed sequence tags (ESTs) was used to characterize potential effector genes. Effectors are small proteins, secreted in planta, that are assumed to facilitate infection. Therefore, a cDNA library was constructed from a compatible interaction. To distinguish pathogen from plant sequences, the library was probed with genomic DNA from V. inaequalis to enrich for pathogen genes, and cDNA libraries were constructed from in vitro-grown material. A suppression subtractive hybridization library enriched for cellophane-induced genes was included, as growth on cellophane may mimic that in planta, with the differentiation of structures resembling those formed during plant colonization. Clustering of ESTs from the in planta and in vitro libraries indicated a fungal origin of the resulting non-redundant sequence. A total of 937 ESTs was classified as putatively fungal, which could be assembled into 633 non-redundant sequences. Sixteen new candidate effector genes were identified from V. inaequalis based on features common to characterized effector genes from filamentous fungi, i.e. they encode a small, novel, cysteine-rich protein, with a putative signal peptide. Three of the 16 candidates, in particular, conformed to most of the protein structural characteristics expected of fungal effectors and showed significant levels of transcriptional up-regulation during in planta growth. In addition to candidate effector genes, this collection of ESTs represents a valuable genomic resource for V. inaequalis.

Two novel Venturia inaequalis genes induced upon morphogenetic differentiation during infection and in vitro growth on cellophane

Venturia inaequalis is a hemibiotrophic ascomycete that causes apple scab. Germ tubes, from conidia or ascospores, penetrate the leaf or fruit surface directly via appressoria-like swellings; subsequently the hyphae divide laterally to form a stroma between the cuticle and the outer wall of the epidermal cells. This morphological switch can be mimicked by growing the fungus in vitro on cellophane discs. The aim of this work was to identify genes upregulated in planta using growth on cellophane as a model. Four cDNA clones were found to be induced by growth on cellophane, and qRT-PCR showed two of these genes were up-regulated over a thousand fold in infected apple leaves compared to liquid culture. The predicted proteins for both genes possess putative signal peptides for secretion but have no similarity to sequences in publicly available databases. Both genes encode proteins with novel, imperfect repeat domain structures, the number of which vary in an isolate-specific fashion. Cin1 has seven or eight repeats of about 60 amino acids with four conserved cysteine residues per repeat, while Cin3 has four or five repeats of 32 amino acids with no cysteines. Both proteins appear to have evolved through internal duplication. Cin3, in particular, shows considerable between-strain variation in domain structure, indicating a high degree of recombination at this locus and revealing that the repeat structure has most likely arisen by unequal crossing-over. Results of this study support the hypothesis that cellophane-grown V. inaequalis mimics aspects of biotrophic infection and provide the first insights into novel fungal genes expressed during apple scab infection and their mechanisms of evolution.

Transcriptome Sequencing Uncovers the Avr5 Avirulence Gene of the Tomato Leaf Mold Pathogen Cladosporium fulvum

The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta-induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization

Significance Anthraquinones are potent secondary metabolites produced by many fungi and plants used in traditional Chinese and Indian medicine. Many display useful biological properties, including antineoplastic, antiinflammatory, antiinfective, or antiparasitic activities. The chemical structure of anthraquinones is very diverse, with many occurring as homo- and heterodimers. Anthraquinone biosynthetic pathways must be elucidated before novel structurally complex chemicals with new or enhanced biological activity can be engineered. In this study, we identified an enzyme involved in asymmetrical dimerization of nataloe-emodin, which results in increased cytotoxicity toward a range of cancer cell lines. Mastering the substrate specificity of this enzyme (and other similar enzymes) could lead to the dimerization of anthraquinone-related compounds with medicinal activities. Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

Repeat-containing protein effectors of plant-associated organisms

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

A conserved proline residue in Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response

CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum

Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr) genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i) frameshift mutations and (ii) transposon insertions in Avr2, (iii) point mutations in Avr4 and Avr4E, and (iv) deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.