Hachiro Oku

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5′-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5′-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.

Participation of toxin in wilting of Japanese pines caused by a nematode

Wilting of Japanese pines (Pinus densifolia and P. thumbergiO, the most serious forest disease in Japan just as the Dutch elm disease in North America, was found recently to be caused by a nematode, Bursaphelenchus lignocolus Mamiya et Kiyohara [1], which is transmitted by a cerambycid beetle, Monochamus alternatus Hope [2]. This disease is spreading epidemically throughout central to south-western part of Japan. In spite of the well established life cycle of the pathogenic nematode [3] and the controlling method of this disease [4], the detailed mechanisms of the rapid wilting of pine trees remained obscure. The pathogenic nematode, B. lignocolus was cultured on the mycelia of Botrytis cinerea grown on the potato sucrose agar medium in Petri dishes at 25 ~ for 2-3 weeks. The pathogenicity of nematode or toxicity of the metabolite was determined by injecting the nematode or 25 gl of the metabolite solutions into the stems of 1month-old pine seedlings which were grown on wet cotton in Petri dishes at 25 ~ under artificial illumination. As shown in Table 1, all pine seedlings wilted within 4 days after inoculation with living nematodes (10 nematodes/seedling).

Molecular cloning of chalcone synthase cDNAs from Pisum sativum.

Chalcone synthase (CHS) is the key regulatory enzyme of flavonoids and isoflavonoids biosynthesis, catalyzing the formation of naringeninchalcone from molecules of three malonyl-CoA and one 4-coumaroyl-CoA in plants [4]. It has been reported that CHS expression is regulated by environmental stimuli such as phytopathogens, elicitors [10, 14] and UV light irradiation [7]. A fungal pathogen of pea, Mycosphaerella pinodes, secretes elicitors and suppressors for the accumulation of the pea isoflavonoid phytoalexin, pisatin, in spore germination fluid [8]. The elicitors induce pisatin accumulation, accompanying with the gene activation encoding CHS and phenylalanine ammonia-lyase (PAL) [14]. Whereas the suppressors have the effects to delay the activation of the pea disease resistance responses such as a 3 hour delay in the accumulation of pal and chs mRNAs [14]. Namely, M. pinodes seems to establish the infection by the pathogenicity factors, suppressors, which inhibits early host active defense responses induced by elicitors. To analyze the mechanisms of gene activation and suppression by the elicitors and suppressors, we first cloned the genes encoding the enzymes for pisatin biosynthetic pathway. Here we report the nucleotide sequences of three chs cDNAs. A c D N A library was constructed from poly(A) + RNA extracted from pea epicotyls 4.5 h after the elicitor treatment by standard procedures. Using a bean chs cDNA, pCH S 1 [10, 11], 36 positive clones were obtained from approximately 5 x 10 4 independent clones as a result of three rounds of screening. It has been reported that CHS is encoded by a small multigene family in pea [6]. Restriction mapping analysis revealed that 36 cDNA clones were grouped into at least five classes. Among them nucleotide sequences of the three cDNAs (pCC6, 2 and 11) representing each class were determined by the dideoxynucleotide sequencing method [ 12] (Fig. 1). Chs-cDNAs of pCC6, 2 and 11 start from 19, 63 and 19 nucleotides upstream of the putative translational start codon, ATG, respectively. They all contain a single open reading frame comprising 389 amino acids (approximately 42 800 Da). The nucleotide sequences and the deduced amino acid sequences in the coding region of the three cDNAs display a homology as high as > 90~o and > 95~o, respectively (Fig. 1 and 2).

Effect of earlier inoculation on the establishment of a subsequent fungus as demonstrated in powdery mildew of barley by a triple inoculation procedure

Abstract The irreversibility of a primary cellular recognition and subsequent conditioning to pathogens were elucidated by a triple inoculation method. Incompatible powdery mildew fungi were not capable of inducing a rejection reaction in leaf areas that had been earlier inoculated with a compatible race, and the growth of non-pathogens in the inoculated area was not inhibited by compatible or non-pathogenic races inoculated as the final challenger. More than 90% of compatible conidia that formed appressoria, after having been applied as the last challenger, were capable of establishing infection in cells which harbored primary haustoria, whereas a large proportion of them failed to infect cells located distantly from the haustoria, irrespective of the presence or absence of a secondarily inoculated non-pathogen. These facts indicate that induced accessibility is localized to a few cells surrounding the primary infection and that the non-pathogen is incapable of initiating the operation of a resistance mechanism in cells within the accessibility-induced area. On barley leaves that had been first inoculated with the wheat mildew fungus (i.e. incompatible), a second compatible race induced accessibility as shown by the apparent increase in ESH frequency of the wheat fungus which was inoculated as the final challenger. This apparent increase in accessibility was interpreted to be due to cell populations that remained unaffected by the primary interaction. These results indicate that the primary recognition by host cells of an invading fungus becomes irreversible once the cells are physiologically conditioned and that this conditioning predestines their responses to fungi that come later.

Suppressor Production as a Key Factor for Fungal Pathogenesis

Plants are endowed with diverse mechanisms that protect them from pathogenic microorganisms. Active defense, including formation of many chemical and physical barriers (e.g., phytoalexin, infection inhibitor, pathogenesis related proteins, lignin, callose, etc.), is considered the main part of the resistance mechanism, because negating of such defense reactions by prior treatment with several metabolic inhibitors or pre-inoculation with compatible fungi allows pathogenic fungi to invade non-host plants. Barriers induced in plant tissues after fungal invasion seem to block penetration, growth, and reproduction of the pathogen. Resistance-inducing substances called inducers or elicitors are released from spores into spore-germination fluid of both pathogenic and nonpathogenic fungi (Hayami et al., 1982; Shiraishi et al., 1978b). As far as the authors know, there is no pathogen that does not produce elicitors. Elicitors from pathogenic fungi are also able to induce resistance in their host.

Transient expression in electroporated pea protoplasts: Elicitor responsiveness of a phenylalanine ammonia-lyase promoter

SummaryHigh yields of viable pea protoplasts were produced from suspension cultured cells and the conditions for the optimum transient expression of the chloramphenicol acetyltransferase (CAT) gene fused to the CaMV 35S promoter after electroporation were investigated. Conditions for elicitor induction of a member of the phenylalanine ammonia-lyase (PAL) gene family in pea was also investigated using a chimeric gene carrying 480 bp of the putative promoter region of gPAL1 connected to bacterial cat gene and nos terminator. CAT activity was considerably induced by the treatment with fungal elicitor (>100 μg/ml glucose equivalent) isolated from Mycosphaerella pinodes, a pea pathogen.

The Role of Indoleacetic Acid Biosynthetic Genes in Tumorigenicity

Various disease symptoms in plants are caused by infection with microorganisms. Among the most well-characterized, on a molecular basis, is hyperplasia. Plant tumors incited by bacterial pathogens include crown gall (Agrobacterium tumefaciens), olive knot (Pseudomonas savastanoi), bacterial witches’ broom (Corynebacterium fascians), bacterial gall of Japanese wisteria (Erwinia milletiae), and others (Nester et al., 1981; Nester et al., 1984; Kemper et al., 1985; Kosuge et al., 1983; Kado, 1984; Okajima et al., 1974). It has been shown that these hyperplastic tissues arise because of hormone imbalances, mainly in indoleacetic acid (IAA) and cytokinin. These imbalances arise in the host plant after bacterial infection.Dedicated to the memory of Dr. Tsune Kosuge (who died on 13 March 1988).

Structure and Mode of Action of Suppressors, Pathogenicity Factors of Pea Pathogen, Mycosphaerella Pinodes

Mycosphaerella pinodes, a pea pathogen has been known to secrete elicitor and suppressor for pisatin biosynthesis into the spore germination fluid, and suppressor counteracts the activity of elicitor. The suppressor is composed of, at least, two components and demonstrated to be not only the inhibitor of pisatin biosynthesis but also the pathogenicity factor of this fungus by suppressing the expression of all defense reactions of host plants temporary. The target of the crude suppressor is proton pump ATPase in plasma membrane of pea plant. The inhibitory activity of membrane ATPase was non-specific in vitro, that is, inhibitory to ATPases of all plant species tested, but specific only to pea in vivo, namely at the tissue level. According to the lead precipitation procedure for ATPase activity, the inhibitory activity was observed at interface between pea and M. pinodes until 6 hr after inoculation, but not between pea and M. ligulicola (non-pathogen). Thus, the suppressor might lower temporary the cell function to defend by inhibition of proton pump ATPase of the host plant.