The observed phenotypes in ΔMoand1 and Double KO mutants suggested possibility that defects of microtubule functions same as homologous genes such as ApsA and ApsB in Aspergillus nidulans or Mcp5 and Mcp6 in Schzoscharromyces pombe. It is well known a fact that mutants are defective in microtubule function often sensitive to microtubule-depolymerizing agents such as Benomyl. We determined resistance to benomyl by culturing onto complement agar media containing 0.5ug/ml concentration of the inhibitor. Indeed, growth of Double KO mutant was more sensitive than wild-type, although less so than ΔMoand1 mutant.
Interestingly, ΔMoand1 showed significantly reduced growth pattern and only 30% growth of ΔMoand1 was restored. From these results, it can be explained that the phenotypes of abnormal nuclear distribution in conidia, hyphae and even appressorium in ΔMoand1 and Double KO mutant caused by defects of microtubules subsequently affecting nuclear migration in development stages.
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Figure 16. Benomyl resistance of wild-type, ΔMoand1 and Double KO mutant.
(A) Each type of strains was cultured on Complement agar media containing the concentration of Benomyl indicated. Plates were incubated for 10 days.
(B) Growth pattern was represented by graph with percentages of growth compared with wild-type.
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Table 1. Oligo sequences used in this study Name Sequence (5’ → 3’)
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DISCUSSTION
Eukaryotes possess a wide variety of conserved nuclear migration systems by microtubule related proteins, but the functions of these proteins were not identified across different species. In the course of our study to determine the molecular mechanism(s) underlying nuclear migration in M.
oryzae, MoAND1 and MoAND2, were identified and characterized. In Aspergillus nidulans, the ApsA and ApsB proteins which are homologous to MoAND1 and MoAND2 in M. oryzae, affect nuclear migration by defecting astral and cytoplasmic microtubules (Fischer & Timberlake, 1995), (Suelmann et al., 1998). Mutations in A.nidulans ApsA lead to curved and thinner microtubules and in ApsB mutation showed reduction of astral microtubules affecting abnormal nuclear migration and positioning. In Saccharomyces cerevisiea, Num1 protein affects nuclei migration. Moreover, microtubule forces are involved in anchoring the nucleus at bud neck and in early stages of nuclear elongation through the neck (Kormanec et al., 1991).
As shown the results before, function of MoAND1 protein could explained if we assume that MoAND1 serves a similar function as ApsA in Aspergillus nidulans. ApsA interacts with MT-plus end localized dynein is activated, it t u gs a t M Ts a n d p u l l s a t t a c h e d n u c l e i . T h e s i t u a t i o n c o ul d
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similar in M. oryzae where MoAND1 could serve as a docking place for microtubules. In MoAND1 deletion mutant, the contact might not be established and nuclear migration was affected. This can be proved by benomyl compounds that promote microtubule depolymerization and reflect the intrinsic stability of cellular microtubules (Cottingham & Hoyt, 1997). In benomyl media, growth of MoAND1 deletion mutant and double KO mutant of MoAND1 and MoAND2 were more severely affected compared with wild-type. This phenomenon usually showed in mutants that are defective in function of microtubules.
Unlike other organisms such as A. nidulas, S. cerevisiae, nuclear migration and positioning is especially important to a plant pathogenic fungus, M.
oryzae for development specialized structure called appressoria. Previous analysis has shown that development of appressoria in the rice blast fungus requires a morphogenetic program regulated by the cAMP response pathway and the Pmk1 MAK kinase cascade in response to the hard, hydrophobic rice leaf surface(Dean, 1997),(Wilson & Talbot, 2009). In response to these signals, a germinating conidium undergoes a single round of mitosis, which is a necessary prerequisite for appressorium differentiation (Veneault-Fourrey et al., 2006).
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We have found that deletion of MoAND1 causes pleotropic phenotypes spanning M. oryzae development: namely conidial morphology, appressorium formation and invasive growth. Each of these features contributes to pathogenic fitness. Normal shape of three-celled conidia affects proper germination development on hydrophobic surface of host cell.
It is conceivable that the abnormal conidial morphology in MoAND1 deletion mutant predominantly blocks subsequent development of conidial germination, because nuclei were absent or abnormally distributed in conidia cells. After conidia germination, successful appressoria formation is essential for pathogenicity. In this study, we showed that MoAND1 deletion mutant and double KO mutant of MoAND1 and MoAND2 are incapable of producing functional appressoria. In MoAND1 deletion mutant and double KO mutant of MoAND1 and MoAND2, conidia formed a germ tube and appressorium developed a second germ tube and appressorium at the same or opposite orientation, and subsequently a third, having total of three appressoria. These results caused by abnormal nuclear distribution at conidia and germ tube in MoAND1 deletion mutant and double KO mutant. In these mutants, only about 30% and 50% appresorria from germinated conidia was produced respectively but penetration to rice surface was unsuccessful.
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MoAND1 deletion mutant showed slower hyphal growth and invasive growth in planta resulting only a few invasive growths successfully moved next cells. Moreover, in double KO mutant, appressoria even penetrate host cell surface resulting significantly reduced pathogenicity.
Together, these results suggest that further studies characterizing MoAND1 and MoAND2 mediated regulation of nuclear movement well help to dissect the evolutionary mechanisms underlying these key processes, potentially leading to novel strategies for crop protection.
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５３ MoAND2 (M. oryzae Abnormal Nuclear Distribution2)라 명명하
였다. MoAND1과 MoAND2의 mutant를 이용한 기능분석에 따르
double KO 군주에서는 모두 microtubules depolyzing Benomyl에 더 민감하게 반응함에 따라 위에서 나타난 비정상적인 핵 이동의 원인이 microtubule의 기능 이상에 기인함을 증명할 수 있었다.
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따라서, 본 연구에서 기능 분석한 두 핵 이동 유전자에 대한 연구 결과는 병 발생에 있어 중요한 역할을 규명한 것으로 판단되어 석 사학위논문으로 충분한 가치가 있다고 여겨진다.
주요어 : 벼 도열병균, 핵 이동 학 번 : 2011-21275