Histone H2B Monoubiquitination Is Involved In Regulating The Dynamics Of Microtubules During The Defense Response To Verticillium Dahliae In Arabidopsis

Histone monoubiquitination is one of histone post-translational modifications which plays an important role in the regulation of genes transcription. Histone H2B monoubiquitination (H2Bub) regulates genes expression, thereby regulates variety of physiological and biochemical processes. H2Bub plays an important role in regulating flowering time, seed germination, root and leaf development and other processes. Recent studies have shown that H2Bub played a key role in plant resistance to fungi.When suffer from pathogen infection, the plant will adopt series of intracellular signal transduction. The microtubules dynamics is an important role in this process. In plant cells, the microtubules dynamics is subject to complex regulations, but the molecular mechanisms of the microtubules dynamics that are involved in responses to biotic stress are largely unknown.In this study, we used wild-type and H2Bub loss of function mutants hub1-4, hub2-2, hub1-4 hub2-2 and ubc1-1 ubc2-1 of Arabidopsis to elucidate which of its mechanisms were involved in plant’s defense response to Verticillium dahliae toxins (Vd toxins), and to reveal physiological and biochemical mechanisms of microtubules dynamics in resistance to Verticillium wilt.After treatment with Verticillium dahliae spores, we found that H2Bub loss of function mutants showed weakened resistance phenotypes compared to wild-type; the number of dead cells of mutants was less than that of wild-type and we verified the cell death induced by Vd toxins was programmed cell death (PCD); in addition, the anthocyanin accumulation of mutants was less than that of wild-type. These results showed that:H2Bub is participates in defense response of Arabidopsis resistant to Vd toxins.To further illustrate the intracellular mechanisms, we used confocal laser scanning microscope (CLSM) to observe the microtubules dynamics of pavement cells after treated with Vd toxins. the results showed that microtubules depolymerization speed became slower, lighter in mutants. We treated Arabidopsis seedlings with tyrosine kinase inhibitor genistein (GN) and tyrosine phosphatase inhibitor phenylarsine oxide (PAO) to investigate the relationship between tyrosine phosphorylation and microtubules dynamics. The results demonstrated that:inhibition of tyrosine kinase activity can stabilize microtubules and inhibition of tyrosine phosphatase activity can promote microtubules depolymerization.we used Real-Time PCR to study the effection on gene expression of H2Bub. The results showed that the expression of tyrosine kinase gene CRK41, tyrosine phosphatase genes PTP1 and PFA-DSP5 were altered in mutants. It was speculated that H2Bub may regulate the dynamics of microtubule cytoskeleton by affecting the level of tyrosine phosphorylation of proteins which regulate the dynamic of microtubule cytoskeleton.Genes expression profiling in wild-type and the mutants were investigated with Vd toxins treatment. It was found that genes of incompatible interaction defense response, defense response, response to bacterium, response to reactive oxygen species, immune system process, cell redox homeostasis, toxin catabolic process, response to biotic stimulus, response to fungus, response to salicylic acid stimulus were up-regulated or inhibited down-regulation in wild-type whereas none or less in the mutants. These results indicate that histone H2B monoubiquitination control many physiological processes associated with plant defense.Taken together, H2Bub influences genes expression such as CRK41, PTP1 and PFA-DSP5 in defense response, thus regulates intracellular protein tyrosine phosphorylation level; tyrosine phosphorylation regulates microtubules dynamics.These results will provide new insight into the mechanisms controlling the Arabidopsis defense response against Vd toxins.