RESUMO
The biogenesis of root-knot nematode (Meloidogyne spp.)-induced galls requires the hyperactivation of the cell cycle with controlled balance of mitotic and endocycle programs to keep its homeostasis. To better understand gall functioning and to develop new control strategies for this pest, it is essential to find out how the plant host cell cycle programs are responding and integrated during the nematode-induced gall formation. This work investigated the spatial localization of a number of gene transcripts involved in the pre-replication complex during DNA replication in galls and report their akin colocation with the cell cycle S-phase regulator Armadillo BTB Arabidopsis Protein 1 (ABAP1). ABAP1 is a negative regulator of pre-replication complex controlling DNA replication of genes involved in control of cell division and proliferation; therefore, its function has been investigated during gall ontogenesis. Functional analysis was performed upon ABAP1 knockdown and overexpression in Arabidopsis thaliana. We detected ABAP1 promoter activity and localized ABAP1 protein in galls during development, and its overexpression displayed significantly reduced gall sizes containing atypical giant cells. Profuse ABAP1 expression also impaired gall induction and hindered nematode reproduction. Remarkably, ABAP1 knockdown likewise negatively affected gall and nematode development, suggesting its involvement in the feeding site homeostasis. Microscopy analysis of cleared and nuclei-stained whole galls revealed that ABAP1 accumulation resulted in aberrant giant cells displaying interconnected nuclei filled with enlarged heterochromatic regions. Also, imbalanced ABAP1 expression caused changes in expression patterns of genes involved in the cell division control as demonstrated by qRT-PCR. CDT1a, CDT1b, CDKA;1, and CYCB1;1 mRNA levels were significantly increased in galls upon ABAP1 overexpression, possibly contributing to the structural changes in galls during nematode infection. Overall, data obtained in galls reinforced the role of ABAP1 controlling DNA replication and mitosis and, consequently, cell proliferation. ABAP1 expression might likely take part of a highly ordered mechanism balancing of cell cycle control to prevent gall expansion. ABAP1 expression might prevent galls to further expand, limiting excessive mitotic activity. Our data strongly suggest that ABAP1 as a unique plant gene is an essential component for cell cycle regulation throughout gall development during nematode infection and is required for feeding site homeostasis.
RESUMO
Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls.
