Valsa mali effector Vm_04797 interacts with adaptor protein MdAP-2ß to manipulate host autophagy.

Apple Valsa canker, caused by the ascomycete fungus Valsa mali, employs virulence effectors to disturb host immunity and poses a substantial threat to the apple industry. However, our understanding of how V. mali effectors regulate host defense responses remains limited. Here, we identified the V. mali effector Vm_04797, which was upregulated during the early infection stage. Vm_04797, a secreted protein, suppressed Inverted formin 1 (INF1)-triggered cell death in Nicotiana benthamiana and performed virulence functions inside plant cells. Vm_04797 deletion mutants showed substantially reduced virulence toward apple. The adaptor protein MdAP-2ß positively regulated apple Valsa canker resistance and was targeted and degraded by Vm_04797 via the ubiquitination pathway. The in vitro analysis suggested that Vm_04797 possesses E3 ubiquitin ligase activity. Further analysis revealed that MdAP-2ß is involved in autophagy by interacting with Malus domestica autophagy protein 16 MdATG16 and promoting its accumulation. By degrading MdAP-2ß, Vm_04797 inhibited autophagic flux, thereby disrupting the defense response mediated by autophagy. Our findings provide insights into the molecular mechanisms employed by the effectors of E3 ubiquitin ligase activity in ascomycete fungi to regulate host immunity.

New Mechanism of Gemcitabine and Its Phosphates: DNA Polymerization Disruption via 3'-5' Exonuclease Inhibition.

Gemcitabine (dFdC), a modified deoxycytidine (dC) widely used in tumor treatment, is a prodrug that is phosphorylated to generate mono-, di-, and triphosphates. The triphosphate (dFdCTP) is incorporated into DNA to terminate DNA synthesis in cancer. Some incorporated dFdC nucleotides can be partially removed by the 3'-5' exonuclease activity, namely its editing function, and the others escape the editing. However, whether there is an active mechanism for dFdC to escape the editing remains unclear. We have first discovered that unlike dFdC, its mono-, di-, and triphosphates can inhibit the 3'-5' exonuclease of DNA polymerase I, suppress editing, and allow the active escaping mechanism, whereas its polymerase activity is not remarkably affected. As such, these phosphates can prevent the removal of the incorporated dFdC residue, thereby actively blocking DNA extension and synthesis. The inhibition efficiency of these phosphates follows the increased order of the mono-, di-, and triphosphates of gemcitabine (dFdC < dFdCMP < dFdCDP < dFdCTP). In addition, after the deletion of the 3'-5' exonuclease of cellular DNA polymerase I, the Escherichia coli mutant is more sensitive to dFdCTP than is wild-type E. coli. Our new discovery of the ability of these dFdC phosphates to inhibit exonuclease activity suggests a novel anticancer mechanism of gemcitabine and its phosphate derivatives.

Synthesis of Selenium-Triphosphates (dNTPαSe) for More Specific DNA Polymerization.

2'-Deoxynucleoside 5'-(alpha-P-seleno)-triphosphates (dNTPαSe) have been conveniently synthesized using a protection-free, one-pot strategy. One of two diastereomers of each dNTPαSe can be efficiently recognized by DNA polymerases, while the other is neither a substrate nor an inhibitor. Furthermore, this Se-atom modification can significantly inhibit non-specific DNA polymerization caused by mis-priming. Se-DNAs amplified with dNTPαSe via polymerase chain reaction have sequences identical to the corresponding native DNA. In conclusion, a simple strategy for more specific DNA polymerization has been established by replacing native dNTPs with dNTPαSe.