AUTHOR=Zheng Wei , Peng Zijia , Peng Shaobing , Yu Zhongdong , Cao Zhimin 

TITLE=Multinuclei Occurred Under Cryopreservation and Enhanced the Pathogenicity of Melampsora larici-populina

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.650902

DOI=10.3389/fmicb.2021.650902

ISSN=1664-302X

ABSTRACT=Melampsora larici-populina is a macrocyclic rust, and the haploid stage with two nuclei and the diploid of mononuclear sequentially occur annually. During the preservation of dry urediniospores at -80 ℃, we found that one isolate, △Ts06, was different from the usual wild-type isolate Ts06 at -20℃ because it has mixed polykaryotic urediniospores. However, the other spores, including the 0, I, III, IV stages of a lifecycle, were the same as Ts06. After five generations of successive inoculation and harvest of urediniospores from the compatible host Populus purdomii, the isolate △Ts06 steadily maintained more than 20% multiple nuclei spores. To test the pathogenesis variation of △Ts06, an assay of host poplars was applied to evaluate the differences between △Ts06 and Ts06. After △Ts06 and Ts06 inoculation, leaves of P. purdomii were used to detect the expression of small secret proteins (SSPs) and fungal biomasses using quantitative real-time PCR (qRT-PCR) and trypan-blue staining. △Ts06 displayed stronger expression of 5 SSPs and had a shorter latent period, a higher density of uredinia and higher DNA mass. A transcriptomic comparison between △Ts06 and Ts06 revealed that 3224 were differentially expressed genes, 55 of which were related to reactive oxygen species metabolism, the MAPK signalling pathway and the meiosis pathway. 10 genes in the mitotic and meiotic pathways and another 2 genes associated with the ‘response to DNA damage stimulus’ all had an upward expression, which were detected by qRT-PCR in △Ts06 during cryopreservation. Gas chromatography-mass spectrometry confirmed that the amounts of Hexadecanoic acid and Octadecadienoic acid were much more in △Ts06 than in Ts06. In addition, using spectrophotometry, Hydrogen Peroxide was also present in greater quantities in △Ts06 compared to those found in Ts06. Increased fatty acids metabolism could prevent damage to urediniospores in super-low temperatures, but oxidant species involved H2O2 may destroy tube proteins of mitosis and meiosis, which could cause abnormal nuclear division and lead to multinucleation that has a different genotype. Therefore, the multinuclear isolate is different from the wild-type isolate in terms of phenotype and genotype; this multinucleation phenomenon in urediniospores improves the pathogenesis and environmental fitness of M. larici-populina.