Plant DNA polymerases α and δ mediate replication of geminiviruses

Abstract

Geminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.

Geminiviruses encode additional small proteins with specific subcellular localizations and virulence function

“…The TYLCV genome contains six known open reading frames (ORFs), encoding the capsid protein (CP)/V1 and V2 in the virion strand, and C1/Rep, C2, C3, and C4 in the complementary strand. Rep creates a cellular environment permissive for viral DNA replication and attracts the DNA replication machinery to the viral genome (reviewed in 8 ); C2 suppresses post-transcriptional gene silencing (PTGS) 9 , protein ubiquitination 10 and jasmonic acid (JA) signaling 10 , 11 ; C3 interacts with PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA), the NAC family transcription factor SlNAC1, and the regulatory subunits of DNA polymerases α and δ to enhance viral replication 12 – 14 ; C4 is a symptom determinant, interferes with the intercellular movement of PTGS, and hampers salicylic acid (SA)-dependent defenses 9 , 15 , 16 ; the CP forms the viral capsid and is essential for the transmission by the insect vector, and shuttles the viral DNA between the nucleus and the cytoplasm 17 – 24 ; V2 is a strong suppressor of PTGS as well as transcriptional gene silencing (TGS), and it mediates the nuclear export of CP 25 – 29 . Interestingly, a recent report identified 21 transcription initiation sites within the TYLCV genome by taking advantage of cap-snatching by rice stripe virus (RSV) in the experimental Solanaceae host Nicotiana benthamiana , suggesting that transcripts beyond those encoding these known ORFs might exist 30 .…”

Mutations in DNA polymerase δ subunit 1 mediate CMD2-type resistance to Cassava Mosaic Geminiviruses

“…This hypothesis is supported by the co-localization of MePOLD1 mutations to those in yeast and humans known to decrease DNA replication activity, and accuracy 40,41,45 . We cannot exclude, however, that the MePOLD1 mutations weaken or block interactions with the virus replication-enhancer protein AC3, which interacts with subunits of POLD 31 . CMD2 resistance has remained robust in farmers' fields over at least two decades.…”

“…Components of the DNA polymerase complex have been reported previously as required for susceptibility to geminiviruses [29][30][31][32][33] . To understand if this holds true for cassava, we targeted MePOLD1 for downregulation in the CMD-susceptible cassava variety 60444 using VIGS (MePOLD1-VIGS) 34 .…”

“…8). A significant reduction in Tomato yellow leaf curl virus (TYLCV) accumulation in Nicotiana benthamiana was also observed after POLD was downregulated by Tobacco rattle virus (TRV)-mediated VIGS 31 . Since TRV is an RNA virus that replicates via a double-strand RNA intermediate, downregulating POLD with TRV-VIGS will not reduce VIGS-mediated siRNA production because TRV is not dependent on POLD for its replication.…”

Small but mighty: Functional landscape of the versatile geminivirus-encoded C4 protein

“…Functional information of geminivirus-encoded proteins is rather fragmentary; nevertheless, the function of the viral proteins encoded by certain positional homologues seems to be frequently conserved across geminiviruses in different genera. That is the case for the replication-associated protein (Rep/C1/AC1), which reprograms the cell cycle and enables replication of the viral DNA, or of REn/C3, which is described to act as a replication enhancer (reviewed in [ 3 , 4 ]). Along the same lines, V2 acts as a silencing suppressor in all geminiviruses tested to date (reviewed in [ 5 ]), and CP forms the viral capsid and acts as the NSP in monopartite geminiviruses (reviewed in [ 3 ]).…”