RAD51 and BRCA2 are conserved proteins involved in homology-directed DNA double-strand break (DSB) repair. This form of DSB repair occurs in somatic cells and is also the essence of meiotic recombination, which allows gametes to have different combinations of alleles and facilitates proper division of chromosomes into haploid gametes. Organisms with rad51 or brca2 mutations often have fertility defects Additionally, there is emerging evidence of a link between DSB repair and plant defense. This dissertation focuses on the RAD51A1, RAD51A2, and BRCA2 proteins in maize. Little is known about the interactions between RAD51A1 and other maize proteins. A series of in vitro affinity experiments were performed: phage display (a discovery-driven molecular biology technique typically used for biomedical applications which finds short amino acid sequences with in vitro affinity for a bait protein) followed by dot-blotting and ELISA to verify affinity. To validate whether these alignments with maize proteins signified possible affinity for RAD51A1, short peptides were synthesized based on the aligning sequences found in maize. Of these 32 synthesized peptides, 14 bound RAD51A1 in vitro, including four peptides that match transcription factors. This is a promising avenue of investigation—in Arabidopsis, RAD51 appears to function as a transcription factor during defense.
Mutants were used to investigate the function of RAD51A1, RAD51A2, and BRCA2 in maize reproduction. Previous evidence of sterility in these mutants relied on the macroscopically visible phenotypes of defective male tassels and low female seed sets. In contrast, this dissertation involved direct examination of reproductive cells. Male sterility was examined by pollen staining. In addition to the expected finding of almost complete sterility in rad51a1/rad51a2 and brca2 mutants, there was an unexpected finding of reduced fertility in rad51a1 single mutants. This reduced male fertility, despite the fact that these plants had one or two wild-type RAD51A2 alleles, suggests that RAD51A1 may have some key reproductive function partially lost in RAD51A2. The basis of the male sterility phenotype was also investigated in male zygotene and pachytene meiocytes. ZYP1, a protein involved in pairing homologous chromosomes, was localized in these cells. As predicted, rad51a1/rad51a2 and brca2 mutants had impaired ZYP1 axis formation compared to wild type; ZYP1 co-localized with DNA but with a more punctate and fragmented appearance compared to wild type. This suggests faulty pairing of chromosomes in these mutants, which could explain their failure to form fertile pollen.
Finally, this thesis discusses transcriptomic and reverse genetics investigations of the potential link between DNA repair and plant defense. Wild-type maize plants were treated with UV-B or salicylic acid (SA), inoculated with Xanthomonas vasicola, or used as a control. RNA-seq data from this experiment showed that the defense genes PR1(PATHOGENESIS-RELATED PROTEIN 1, or Zm00001d018738) and PRP3 (PATHOGENESIS-RELATED PROTEIN 3, or Zm00001d048947) had increased transcription in inoculated samples. There was also increased transcription of the Bowman-Birk type trypsin inhibitor Zm00001d024960. This experiment also uncovered several genes of interest involved in maize response to UV, including genes involved in diterpenoid metabolism. Only one gene met the adjusted p-value (padj) <0.1 threshold for significant upregulation in both UV- and SA-treated maize, the fatty acid biosynthesis gene ECERIFERUM 1 (Zm00001d014055). In a reverse genetics experiment on the possible role of BRCA2 in defense, brca2 mutants were inoculated with X. vasicola. Loss of this gene was found to affect lesion length in the opposite direction from what had been hypothesized--brca2 mutants had 3.4 cm shorter lesions (p= 7.45E-05). This result was in contrast with a previous finding from Arabidopsis—brca2a Arabidopsis mutants have increased susceptibility to Pseudomonas syringae. This unexpected finding led to two hypotheses to explain the shorter lesions in the maize mutants: either the brca2 mutants are less susceptible and less X. vasicola grows in these plants’ leaves, or the brca2 mutants have impaired response to X. vasicola, leading to a reduced ability of the plant to mount a defense response and create the lesion. A second experiment showed weak evidence for the hypothesis that the brca2 mutants had less X. vasicola in their leaf tissue (p= 0.155). However, there was no evidence that brca2 mutants had impaired response to X. vasicola—PR1 expression was, if anything, slightly higher in the mutants (p=0.533). Considered together, these results suggest a role for BRCA2 in X. vasicola susceptibility and response that is worth further investigation.