| Pine wilt disease (PWD) is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, which is believed to be the most destructive diseases to pine tree species, resulting in economic and ecological loss in the global scope. PWD is the multiple complex disease systems, its pathogenic mechanism has been controversial. At present, analysis of pine’s pathological response to related molecular mechanisms after PWN invasion has become an important way to conquer the pathogenesis of PWN and disease resistance of pine trees. Signal transduction is the initial event of the resistance reaction in plants, Ca2+as an important intracellular second messenger, plays an important role in signal transduction of plant defense response. In the study, we did a set of work on the calcium signal transduction characteristics of pine response to PWD infection. The results is as followed:The experiment system study on the interactions of Pinus massoniana and B. xylophilus was established: high virulence PWN strain NXY61was obtained by separation, identification and screening in the study. The migration rule of B. xylophilus in P. massoniana followed time development was determined. The result would provide the foundation for studing calcium signal transduction characteristics in pine in the early of PWD infection.Extracellular Ca2+flux profiles in different organs of Masson pine30-180min after B. xylophilus NXY61inoculation was observed by non-invasive micro-test technique (NMT). This study confirms that specific calcium transient and calcium oscillation characteristics appear in masson pine root, stem and leaf in the early of PWN infection; and the calcium transient and calcium oscillation characteristics have significant differences among different organs. Calcium transient characteristics is as followed:transient Ca2+inflow in masson pine root, stem and leave happens in the early of PWN infection and significant changes of Ca2+inflow velocity started to appear in15min,7min,7min after inoculation, respectively; the respective maximum velocity of Ca2+inflow is root (3700pmolcm-2s-1)> leaf (1300pmolcm-2s-1)> stem (250pmolcm-2s-1), and the respective duration of Ca2+inflow is stem (more than48min)> leave (43min)> root (20min). And the calcium oscillation characteristics is as followed: before velocity of Ca2+inflow shows significant changes after PWN infection, the amplitude and frequency of calcium oscillation in masson pine root, stem and leaf does not change significantly; while velocity of Ca2+inflow reduces to the stationary stage, the frequency of calcium oscillation does not change significantly, but the amplitude is significantly reduced in stem, significantly increased in leaf, and amplitude in leaf is significantly greater than that in stem. In a word, this study reveals that the Ca2+change in masson pine is response to PWN infection, and specific calcium signals produce in masson pine root, stem and leaf due to the PWN infection.In the study, based on the bioinformatics methods and the constructed four subtractive suppression hybridization cDNA libraries (SSH) by taking time-course samples from PWN-inoculated Masson pine trees, we studied the molecular characteristics and signal transduction characteristics of pine early after PWD infection. One-hundred forty-four differential expression sequence tags (ESTs) were in-depth analysis. It is found that:in the early stage of pine wood nematode infection (24h,72h), lots of calcium related protein genes expression are induced, including EFh family proteins, calreticulin, and prediction calmodulin-binding protein (CaMBPs). Combined with bioinformatics analysis as well as the previous reported studies,6types of CaMBPs,which exercise different functions in the early stage of PWN infection, including heat shock protein HSP70, HSP101, pathogenesis related protein PR-14and Bcl-2family protein BAG6participate in defense response process; aldehyde enzyme I is involved in the detoxification and redox process; NPG1is involved in the signal transduction process. In addition, based on SSH, It is found that: in the early stage of pine wood nematode infection (24h,72h), a lot of defense related protein genes expression are induced, including encoding receptor resistant protein RPS, pathogenesis related protein PR-3, PR-4, PR-5, PR-14and flavonoid synthesis related CHS gene; at the same time, lignin biosynthesis related protein genes including methionine synthase expression2, PCBER, CAD, CoMT, CCoAOMT and dirigen-like protein genes are all up-regulated expression; GPX and Cyt_b561FRRS1like-containing protein genes participating in the reduction process of H2O2and AER participating in catalytic reduction process of activated carbon are also upregulated expression. The results of this study reveals that early during pine wood nematode infection, masson pine got especial interactions characteristics, and massive calcium related protein genes, including6kinds of CaMBP genes, are induced expression early after PWN infection, suggesting that a important calcium signal transduction pathway mediated by CaMBPs may exist, and involves in regulation of masson pine defense response.Calmodulin (CaM) is the key node protein locating upstream CaMBPs, and CaM, as the main receptor protein Ca2+, plays an important role in the transmission of Ca2+signals, it may transmit Ca2+signaling via CaMBPs. Based on the ordinary PCR and RACE technology, combined with the predicted protein structure and phylogenetic tree analysis, CaM and CaMBPs are approved to be in pine and it may transmit Ca2+signaling via CaM. in our study, CaM gene (pmCaM) was cloned from masson pine for the first time; and calcium binding protein genes pmCBP100, pmCBP’153and CaMBP gene (pmCaMBP), which are response to PWN infection, were also cloned from masson pine in this study. Among them, pmCBP100, pmCBP153and pmCaM belonged to EFh family protein, had EFh domains, directly combining with Ca2+While pmCaMBP is a kind of transmembrane protein, has CaMBD and TPR domains. Its CaMBD located543-554amino acid position in the protein; three TPRs located428-509,573-673,606-707amino acid positions in the protein, and the function of TPRs is involved in protein-protein interaction for cell signal transduction in plant.Based on Real time fluorescence quantitative PCR technology, expression pattern of pmCaM and pmCaMBP in half a year old masson pine seedlings30min,45min,60min,90min,180min and12h,24h,48h,72h,96h after inoculation with strain NXY61was studied. The results showed that after PWN inoculation, the expression of pmCaM, pmCaMBP is significantly changed; and the time points for significant changes and the change trend and degree at the same point of CaM, CaMBP expression are different among organs. In a word, this study reveales that in the early of PWN infection, the pmCaM, pmCaMBP expression of masson pine are in response to PWN infection, and have specific temporal and spatial expression characteristics. Combining with the confirmed calcium signal generated in PWN infection aove, it is confirmed that pmCaM and pmCaMBP participated in the regulation of calcium signaling transmission. In conclusion, the results of this study reveales that the "Ca2+-CaM-CaMBP" signal transduction pathway may exist, participating in the regulation of calcium signaling transmission and involve in the regulation of the early defense responses in pine early after PWN infection. |
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