Kazuya Akimitsu

Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri but not brown spot caused by Alternaria alternata.

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.

Characterization of a hydroperoxide lyase gene and effect of C6-volatiles on expression of genes of the oxylipin metabolism in Citrus.

A number of C6-volatile products of the lipoxygenase (LOX) pathway was examined for their antifungal activity and a potential role as a signal molecule in citrus. trans-2-Hexenal induced the rough lemon lipoxygenase gene (RlemLOX), hydroperoxide lyase gene (RlemHPL) and AOS gene, but hexanal, and hexanol suppressed them. cis-3-Hexenol and trans-2-hexenol increased expression of the AOS gene but not RlemLOX and RlemHPL. Transcripts of the RlemHPL and AOS gene were detected constitutively in leaves by northern blot, but wounding or inoculation with nonpathogenic Alternaria alternata rapidly increased the transcript accumulation. Transcripts of the RlemHPL and AOS genes were also induced with pathogenic A. alternata, which produces the host-selective ACR-toxin, but the signal declined rapidly after inoculation. An increase in enzymatic activity of HPL after wounding or inoculation with nonpathogen was suppressed in leaves infected with the pathogen. Interestingly, vapor treatment with trans-2-hexenol delayed necrotic spot formation in the leaves inoculated with the pathogenic A. alternata. Since trans-2-hexenol has no antifungal activity to A. alternata and also did not inhibit necrosis formation by ACR-toxin alone, the delay of symptoms may be caused by activation of AOS in the LOX pathway to produce oxylipin derivatives such as methyl jasmonate for activation of defense related genes with antifungal activity.

Molecular, ecological and evolutionary approaches to understanding Alternaria diseases of citrus

SUMMARY Alternaria fungi cause four different diseases of citrus: Alternaria brown spot of tangerines, Alternaria leaf spot of rough lemon, Alternaria black rot of several citrus fruits and Mancha foliar of Mexican lime. The first three diseases are caused by the small-spored species, Alternaria alternata and the causal agents can only be differentiated using pathogenicity tests, toxin assays or genetic markers. Mancha foliar is caused by the morphologically distinct, large-spored species A. limicola. Substantial progress has been made in understanding the biology, ecology, population biology, systematics, molecular biology and biochemistry of the interactions between these pathogens and citrus. Epidemiological studies have focused on brown spot of tangerines and their hybrids and have contributed to the development of a model of disease development which has improved control and reduced fungicide use. Studies of the population genetics, host specificity and ecology of A. alternata from different ecological niches on citrus have revealed host specific forms of the pathogen which cause disease on different citrus species, the existence of three phylogenetic lineages of the fungus which cause brown spot world-wide, and closely related non-pathogenic isolates which colonize healthy citrus tissue. The role of host-specific toxins in Alternaria diseases of citrus has been extensively studied for over 20 years, and these pathosystems have become model systems for host-pathogen interactions. Recent molecular research has started to unravel the genetic basis of toxin production and the host susceptibility to toxin, and the role of extracellular, degradative enzymes in disease.

Worldwide Phylogeography of the Citrus Brown Spot Pathogen, Alternaria alternata

ABSTRACT Sixty-five isolates of Alternaria alternata were sampled from brown spot lesions on tangerines and mandarins (Citrus reticulata) and tangerine x grapefruit (C. reticulata x C. paradisi) hybrids in the United States, Colombia, Australia, Turkey, South Africa, and Israel to investigate the worldwide phylogeography of the fungus. Genetic variation was scored at 15 putative random amplified polymorphic DNA (RAPD) loci and 465 bp of an endo-polygalacturonase (endo-PG) gene was sequenced for each isolate. Cluster analysis of RAPD genotypes revealed significant differentiation between United State and Colombia isolates and Turkey, South Africa, Israel, and Australia isolates. Sequencing of endo-PG revealed 21 variable sites when the outgroup A. gaisen (AK-toxin-producing pathogen of Japanese pear) was included and 13 variable sites among the sampled isolates. Nucleotide substitutions at 10 of 13 variable sites represented silent mutations when endo-PG was translated in frame. Eight distinct endo-PG haplotypes were found among the sampled isolates and estimation of a phylogeny with endo-PG sequence data revealed three clades, each with strong bootstrap support. The most basal clade (clade 1) was inferred based on its similarity to the outgroup A. gaisen and consisted exclusively of pathogenic isolates from the United States and Colombia. Clade 2 consisted of pathogenic and nonpathogenic isolates from the United States, Australia, South Africa, and Israel and clade 3 contained pathogenic and nonpathogenic isolates from Australia, South Africa, Israel, and Turkey. Quantitative estimates of virulence (disease incidence) were obtained for isolates from the United States, Colombia, South Africa, Israel, and Turkey by spray inoculating detached citrus leaves and counting the number of lesions 24 h after inoculation. Large differences in virulence were detected among isolates within each location and isolates from the United States were significantly more virulent than isolates from other locations. Several isolates from Colombia, South Africa, Israel, and Turkey had low virulence and 8% of all isolates were nonpathogenic. All but one of the nonpathogenic isolates were found in clade 2 of the endo-PG phylogeny, which also included the most highly virulent isolates sampled.

Distribution and Characterization of AKT Homologs in the Tangerine Pathotype of Alternaria alternata

ABSTRACT The tangerine pathotype of Alternaria alternata produces a host-selective toxin (HST), known as ACT-toxin, and causes Alternaria brown spot disease of citrus. The structure of ACT-toxin is closely related to AK- and AF-toxins, which are HSTs produced by the Japanese pear and strawberry pathotypes of A. alternata, respectively. AC-, AK-, and AF-toxins are chemically similar and share a 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Two genes controlling AK-toxin biosynthesis (AKT1 and AKT2) were recently cloned from the Japanese pear pathotype of A. alternata. Portions of these genes were used as heterologous probes in Southern blots, that detected homologs in 13 isolates of A. alternata tangerine pathotype from Minneola tangelo in Florida. Partial sequencing of the homologs in one of these isolates demonstrated high sequence similarity to AKT1 (89.8%) and to AKT2 (90.7%). AKT homologs were not detected in nine isolates of A. alternata from rough lemon, six isolates of nonpathogenic A. alternata, and one isolate of A. citri that causes citrus black rot. The presence of homologs in the Minneola isolates and not in the rough lemon isolates, nonpathogens or black rot isolates, correlates perfectly to pathogenicity on Iyo tangerine and ACT-toxin production. Functionality of the homologs was demonstrated by detection of transcripts using reverse transcription-polymerase chain reaction (RT-PCR) in total RNA of the tangerine pathotype of A. alternata. The high sequence similarity of AKT and AKT homologs in the tangerine patho-type, combined with the structural similarity of AK-toxin and ACT-toxin, may indicate that these homologs are involved in the biosynthesis of the decatrienoic acid moiety of ACT-toxin.

Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism

The filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host‐specific toxins and cause diseases on different plants. The strawberry pathotype produces host‐specific AF‐toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK‐toxin. The strawberry pathotype produces two related molecular species, AF‐toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT‐1 from the strawberry pathotype that contains three genes involved in AF‐toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT‐1. AFTS1 encodes a protein with similarity to enzymes of the aldo‐ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: ΔaftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF‐toxin II, but not AF‐toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.

Involvement of OsJAZ8 in Jasmonate-Induced Resistance to Bacterial Blight in Rice

The plant hormone jasmonic acid (JA) has a crucial role in both host immunity and development in plants. Here, we report the importance of JA signaling in the defense system of rice. Exogenous application of JA conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) in rice. Expression of OsJAZ8, a rice jasmonate ZIM-domain protein, was highly up-regulated by JA. OsJAZ8 interacted with a putative OsCOI1, which is a component of the SCF(COI1) E3 ubiquitin ligase complex, in a coronatine-dependent manner. OsJAZ8 also formed heterodimers with other OsJAZ proteins but did not form homodimer. JA treatment caused OsJAZ8 degradation and this degradation was dependent on the 26S proteasome pathway. Furthermore, the JA-dependent OsJAZ8 degradation was mediated by the Jas domain. Transgenic rice plants overexpressing OsJAZ8ΔC, which lacks the Jas domain, exhibited a JA-insensitive phenotype. A large-scale analysis using a rice DNA microarray revealed that overexpression of OsJAZ8ΔC altered the expression of JA-responsive genes, including defense-related genes, in rice. Furthermore, OsJAZ8ΔC negatively regulated the JA-induced resistance to Xoo in rice. On the basis of these data, we conclude that JA plays an important role in resistance to Xoo, and OsJAZ8 acts as a repressor of JA signaling in rice.

Characterization of cDNAs Encoding Two Distinct Miraculin-like Proteins and Stress-related Modulation of the Corresponding mRNAs in Citrus jambhiri Lush

Two distinct full-length cDNAs from rough lemon that encoded miraculin-like proteins were isolated by random amplification of cDNA ends (RACEs), based on sequence information from subtractive PCR previously described, and designated as RlemMLP1 and RlemMLP2. The transcripts of both RlemMLP1 and RlemMLP2 were not detected in leaves, or stems but accumulated in fruits. Transcripts accumulated to higher levels in leaves after wounding, inoculation with conidia of Alternaria alternata, or treatment with methyl jasmonate vapors. Treatment with methyl salicylate antagonized the signaling pathway of wounding. Treatment with methyl salicylate at 2 h after wounding significantly reduced wounding-induced gene expression of both RlemMLP1 and RlemMLP2. Protein products of these genes were obtained by using a prokaryotic expression system, and had protease inhibitor activity. RlemMLP2, but not RlemMLP1, contained a thaumatin motif, and only RlemMLP2 showed anti-fungal activity against Alternaria citri. Cellular localization analysis with RlemMLP1 or RlemMLP2 fused to a green fluorescence protein gene following transient translation using a particle bombardment in onion cells indicated that both RlemMLP1 and RlemMLP2 were localized to the cytosol. These evidences revealed that rough lemon RlemMLPs are likely to have defensive function against pathogens at least when host cells are broken by their infections.

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins.

ABSTRACT Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.

Contribution of Peroxisomes to Secondary Metabolism and Pathogenicity in the Fungal Plant Pathogen Alternaria alternata

ABSTRACT The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1, AKT2, AKT3, and AKTR, involved in AK toxin biosynthesis. AKT1, AKT2, and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated AaPEX6, which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made AaPEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAaPEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAaPEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAaPEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata. We also observed that mutation of AaPEX6 caused a marked reduction of conidiation.