Responsive mechanisms of the Japanese white birch (Betula platyphylla Sukaczev var. japonica) plantlet infected with a canker-rot fungus Inonotus obliquus

Chapter 1 Introduction Japanese white birch (Betula platyphylla Sukaczev var. japonica (Miq.) H. Hara) is distributed through subalpine zone in Honsyu and Hokkaido, Japan. Its wood is used for furniture, interior, raw materials of pulpwood, fiberboards, particleboards, and others. On the other hand, Inonotus obliquus (pers. Ex Fr.) Pilat is distributed in Europe, Siberia, China, Japan, and North America. This fungus causes stem heart rot of birch trees, and produces a black solid sclerotium called as sterile conk or canker-like body. Therefore, to prevent Japanese white birch trees from the cancer-rot disease caused by I. obliquus, the responsive mechanisms of the Japanese white birch plantlet infected with I. obliquus should be clarified. The objectives of the present study are to clarify the responsive mechanisms of Japanese white birch plantlets infected with I. obliquus. Three-month-old Japanese white birch plantlets No.8, which were prepared through the in vitro micropropagation techniques, were infected with I. obliquus strain IO-U1. After infection, time-course changes of accumulation of specific phenolic compounds, specific peroxidase (POD) distribution, and POD isozyme expression were analyzed. In addition, specifically produced proteins were also identified by proteome analysis. Based on the obtained results, the responsive mechanisms of the Japanese white birch plantlet infected with a canker-rot fungus I. obliquus were discussed. Chapter 2 Histochemical studies of specific phenolics accumulation in the Japanese white birch plantlet infected with Inonotus obliquus Accumulation of specific phenolic compounds was histochemically observed in the intact (C1), wounded (C2), and infected (T) plantlets. To clarify the time-course changes of the substances, the samples were collected at 2 h to 30 d after the treatments. In the C1 plantlet, no specific autofluorescence of specific phenolic compounds was recognized. On the other hand, the specific autofluorescence of phenolics was observed near the wounded zone of the C2 plantlet. The specific autofluorescence of phenolic compounds in the C2 plantlet was first observed in the cortex after 4 h and in the pith and vessel at 6 h after the wounding. After 10 and 30 d after the wounding, the wound-induced callus developed, and the outer layer of the callus exhibited the specific autofluorescence of phenolic compounds. In addition, specific phenolic compounds were observed in cortical layer, cambium, and callus at 1, 10, and 30 d after wounding, respectively. On the other hand, in the T plantlet, the specific autofluorescence was also observed in the vessel wall at 2 h post-infection, in cortex and cambium at 1 d post-infection, and in pith at 10 d post-infection. In addition, the outer layer of wound-induced callus also showed the specific autofluorescence at 30 d post-infection. From these obtained results, it is considered that massive induction for producing specific phenolic compounds was triggered by fungal infection in Japanese white birch plantlet No.8. Chapter 3 The relationships between distribution of specific peroxidase activity and expression of peroxidase isozyme Time-course changes of the in situ peroxidase (POD) distribution and expression of POD isozymes were investigated for Japanese white birch plantlet No.8 infected with I. obliquus strain IO-U1. The C1, C2, and T plantlets were obtained at 2 h up to 30 d. In situ specific POD activity was detected in the C2 and T plantlets, and the specific POD activity in the T plantlet was more widely distributed compared to that in the C2 plantlet. In addition, the area of the specific POD activity localization was almost the same as that of specific phenolic compounds, although a time lag was found between the appearance of the specific POD activity and the specific phenolic compounds. The POD isozymes were clearly detected within the basic range (pI > 8.5) in isoelectric focusing electropherograms. The activity of cationic POD isozymes in the C2 and T plantlets was induced strongly compared to that in the C1 plantlet. In addition, the pattern of time-course changes in the activities of in situ specific POD and POD isozymes was different between the C2 and T plantlets, suggesting that the responsive mechanisms to fungal infection are different from those to wounding. The obtained results suggest that cationic POD isozymes are related to the responsive mechanisms in Japanese white birch plantlet No.8 against the infection with I. obliquus strain IO-U1. Chapter 4 Proteome analysis of inducible proteins by fungal infection in Japanese white birch plantlet No.8 The proteins produced specifically in Japanese white birch plantlet No.8 by the infection with I. obliquus strain IO-U1 were identified by proteome analysis. The sterile Japanese white birch plantlets were infected with the fungus, and the protein samples obtained at 2 d post-infection were subjected to two-dimensional electrophoresis to detect the infection-specific proteins. The specific proteins were analyzed using MALDI/TOF/MS and identified by peptide mass fingerprinting with MASCOT software. Among the 735 protein spots detected in the infected plantlet, 169 spots were recognized as infection-specific proteins. Of these spots, 91 spots were analyzed using MALDI/TOF/MS, resulted in the identification of two heat shock proteins (Hsp70 and Hsp60) as the infection-specific proteins. Hsp70 and Hsp60 may cooperate to refold the proteins which were denatured by the infection of I. obliquus strain IO-U1 in the birch plantlet. It is suggested that these proteins are expressed in Japanese white birch plantlet No.8 by the stress caused by the infection with I. obliquus strain IO-U1. Chapter 5 Conclusion Based on the results obtained in the present study, the specific POD was expressed on the surface of the plant cell, immediately after the invasion of I. obliquus IO-U1 and the detection by Japanese white birch plantlet No.8. The specific POD is considered to be involved in the H2O2 generation and phenolic oxidation. The specific POD-mediated polymerization of phenolics was activated by the fungal infection. Hsp60 and Hsp70 work for refolding the proteins which were denatured by H2O2 and unidentified fungal effectors. In conclusion, it is considered that Japanese white birch plantlet functionally activates its based defense mechanisms against the infection of I. obliquus, and protects its biological function and physiological activity by repairing the denatured proteins in the plantlet cells. However, the unidentified fungal effectors and suppressors are considered to decrease the basal defense level in the birch plantlet, eventually resulted in the completion of fungal infection and mycelial growth in the birch plantlet.