Data analysis of polygalacturonase inhibiting proteins (PGIPs) from agriculturally important proteomes.

The plant cell wall structure can be altered by pathogen-secreted polygalacturonases (PGs) that cleave the α-(1→4) linkages occurring between D-galacturonic acid residues in homogalacturonan. The activity of the PGs leads to cell wall maceration, facilitating infection. Plant PG inhibiting proteins (PGIPs) impede pathogen PGs, impairing infection and leading to the ability of the plant to resist infection. Analyses show the Glycine max PGIP11 (GmPGIP11) is expressed within a root cell that is parasitized by the pathogenic nematode Heterodera glycines, the soybean cyst nematode (SCN), but while undergoing a defence response that leads to its demise. Transgenic experiments show GmPGIP11 overexpression leads to a successful defence response, while the overexpression of a related G. max PGIP, GmPGIP1 does not, indicating a level of specificity. The analyses presented here have identified PGIPs from 51 additional studied proteomes, many of agricultural importance. The analyses include the computational identification of signal peptides and their cleavage sites, O-, and N-glycosylation. Artificial intelligence analyses determine the location where the processed protein localize. The identified PGIPs are presented as a tool base from which functional transgenics can be performed to determine whether they may have a role in plant-pathogen interactions.

The central circadian regulator CCA1 functions in Glycine max during defense to a root pathogen, regulating the expression of genes acting in effector triggered immunity (ETI) and cell wall metabolism.

Expression of the central circadian oscillator components CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), TIMING OF CAB1 (TOC1), GIGANTEA (GI), and CONSTANS (CO) occurs in Glycine max root cells (syncytia) parasitized by the nematode Heterodera glycines while undergoing resistance, indicating a defense role. GmCCA1-1 relative transcript abundance (RTA) in roots experiencing overexpression (OE) or RNA interference (RNAi) is characterized by rhythmic oscillations, compared to a ribosomal protein gene (GmRPS21) control. A GmCCA1-1 RTA change, advancing by 12 h, exists in H. glycines-infected as compared to uninfected controls in wild-type, H. glycines-resistant, G. max[Peking/PI 548402]. The G. max[Peking/PI 548402] transgenic controls exhibit the RTA change by 4 h post infection (hpi), not consistently occurring in the H. glycines-susceptible G. max[Williams 82/PI 518671] until 56 hpi. GmCCA1-1 expression is observed to be reduced in H. glycines-infected GmCCA1-1-OE roots as compared to non-infected transgenic roots with no significant change observed among RNAi roots. The GmCCA1-1 expression in transgenic GmCCA1-1-OE roots remains higher than control and RNAi roots. Decreased GmCCA1-1 mRNA among infected roots shows the altered expression is targeted by H. glycines. Gene expression of proven defense genes including 9 different mitogen activated protein kinases (GmMAPKs), NON-RACE SPECIFIC DISEASE RESISTANCE 1 (GmNDR1-1), RPM1-INTERACTING PROTEIN 4 (GmRIN4-4), and the secreted xyloglucan endotransglycosylase/hydrolase 43 (GmXTH43) in GmCCA1-1-OE and GmCCA1-1-RNAi roots, compared to controls, reveal a significant role of GmCCA1-1 expression in roots undergoing defense to H. glycines parasitism. The observation that GmCCA1-1 regulates GmXTH43 expression links the central circadian oscillator to the functionality of the secretion system.

Xyloglucan endotransglycosylase/hydrolase increases tightly-bound xyloglucan and chain number but decreases chain length contributing to the defense response that Glycine max has to Heterodera glycines.

The Glycine max xyloglucan endotransglycosylase/hydrolase (EC 2.4.1.207), GmXTH43, has been identified through RNA sequencing of RNA isolated through laser microdissection of Heterodera glycines-parasitized root cells (syncytia) undergoing the process of defense. Experiments reveal that genetically increasing XTH43 transcript abundance in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] decreases parasitism. Experiments presented here show decreasing XTH43 transcript abundance through RNA interference (RNAi) in the H. glycines-resistant G. max[Peking/PI 548402] increases susceptibility, but it is unclear what role XTH43 performs. The experiments presented here show XTH43 overexpression decreases the relative length of xyloglucan (XyG) chains, however, there is an increase in the amount of those shorter chains. In contrast, XTH43 RNAi increases XyG chain length. The experiments show that XTH43 has the capability to function, when increased in its expression, to limit XyG chain extension. This outcome would likely impair the ability of the cell wall to expand. Consequently, XTH43 could provide an enzymatically-driven capability to the cell that would allow it to limit the ability of parasitic nematodes like H. glycines to develop a feeding structure that, otherwise, would facilitate parasitism. The experiments presented here provide experimentally-based proof that XTHs can function in ways that could be viewed as being able to limit the expansion of the cell wall.

Mitogen activated protein kinase (MAPK)-regulated genes with predicted signal peptides function in the Glycine max defense response to the root pathogenic nematode Heterodera glycines.

Glycine max has 32 mitogen activated protein kinases (MAPKs), nine of them exhibiting defense functions (defense MAPKs) to the plant parasitic nematode Heterodera glycines. RNA seq analyses of transgenic G. max lines overexpressing (OE) each defense MAPK has led to the identification of 309 genes that are increased in their relative transcript abundance by all 9 defense MAPKs. Here, 71 of those genes are shown to also have measurable amounts of transcript in H. glycines-induced nurse cells (syncytia) produced in the root that are undergoing a defense response. The 71 genes have been grouped into 7 types, based on their expression profile. Among the 71 genes are 8 putatively-secreted proteins that include a galactose mutarotase-like protein, pollen Ole e 1 allergen and extensin protein, endomembrane protein 70 protein, O-glycosyl hydrolase 17 protein, glycosyl hydrolase 32 protein, FASCICLIN-like arabinogalactan protein 17 precursor, secreted peroxidase and a pathogenesis-related thaumatin protein. Functional transgenic analyses of all 8 of these candidate defense genes that employ their overexpression and RNA interference (RNAi) demonstrate they have a role in defense. Overexpression experiments that increase the relative transcript abundance of the candidate defense gene reduces the ability that the plant parasitic nematode Heterodera glycines has in completing its life cycle while, in contrast, RNAi of these genes leads to an increase in parasitism. The results provide a genomic analysis of the importance of MAPK signaling in relation to the secretion apparatus during the defense process defense in the G. max-H. glycines pathosystem and identify additional targets for future studies.

The heterologous expression of a soybean (Glycine max) xyloglucan endotransglycosylase/hydrolase (XTH) in cotton (Gossypium hirsutum) suppresses parasitism by the root knot nematode Meloidogyne incognita.

A Glycine max (soybean) hemicellulose modifying gene, xyloglucan endotransglycoslase/hydrolase (XTH43), has been identified as being expressed within a nurse cell known as a syncytium developing within the soybean root undergoing the process of defense to infection by the parasitic nematode, Heterodera glycines. The highly effective nature of XTH43 overexpression in suppressing H. glycines parasitism in soybean has led to experiments examining whether the heterologous expression of XTH43 in Gossypium hirsutum (upland cotton) could impair the parasitism of Meloidogyne incognita, that form a different type of nurse cell called a giant cell that is enclosed within a swollen root structure called a gall. The heterologous transgenic expression of XTH43 in cotton resulted in an 18% decrease in the number of galls, 70% decrease in egg masses, 64% decrease in egg production and a 97% decrease in second stage juvenile (J2) production as compared to transgenic controls. The heterologous XTH43 expression does not significantly affect root mass. The results demonstrate XTH43 expression functions effectively in impairing the development of M. incognita at numerous life cycle stages occurring within the cotton root. The experiments reveal that there are highly conserved aspects of the defense response of G. max that can function effectively in G. hirsutum to impair M. incognita having a different method of parasitism.

The tumor suppressor BRCA1-BARD1 complex localizes to the synaptonemal complex and regulates recombination under meiotic dysfunction in Caenorhabditis elegans.

Breast cancer susceptibility gene 1 (BRCA1) and binding partner BRCA1-associated RING domain protein 1 (BARD1) form an essential E3 ubiquitin ligase important for DNA damage repair and homologous recombination. The Caenorhabditis elegans orthologs, BRC-1 and BRD-1, also function in DNA damage repair, homologous recombination, as well as in meiosis. Using functional GFP fusions we show that in mitotically-dividing germ cells BRC-1 and BRD-1 are nucleoplasmic with enrichment at foci that partially overlap with the recombinase RAD-51. Co-localization with RAD-51 is enhanced under replication stress. As cells enter meiosis, BRC-1-BRD-1 remains nucleoplasmic and in foci, and beginning in mid-pachytene the complex co-localizes with the synaptonemal complex. Following establishment of the single asymmetrically positioned crossover on each chromosome pair, BRC-1-BRD-1 concentrates to the short arm of the bivalent. Localization dependencies reveal that BRC-1 and BRD-1 are interdependent and the complex fails to properly localize in both meiotic recombination and chromosome synapsis mutants. Consistent with a role for BRC-1-BRD-1 in meiotic recombination in the context of the synaptonemal complex, inactivation of BRC-1 or BRD-1 enhances the embryonic lethality of mutants defective in chromosome synapsis. Our data suggest that under meiotic dysfunction, BRC-1-BRD-1 stabilizes the RAD-51 filament and alters the recombination landscape; these two functions can be genetically separated from BRC-1-BRD-1's role in the DNA damage response. Together, we propose that BRC-1-BRD-1 serves a checkpoint function at the synaptonemal complex where it monitors and modulates meiotic recombination.

LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining.

The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.

DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

The spindle assembly checkpoint: More than just keeping track of the spindle.

Genome stability is essential for cell proliferation and survival. Consequently, genome integrity is monitored by two major checkpoints, the DNA damage response (DDR) and the spindle assembly checkpoint (SAC). The DDR monitors DNA lesions in G1, S, and G2 stages of the cell cycle and the SAC ensures proper chromosome segregation in M phase. There have been extensive studies characterizing the roles of these checkpoints in response to the processes for which they are named; however, emerging evidence suggests significant crosstalk between the checkpoints. Here we review recent findings demonstrating overlapping roles for the SAC and DDR in metaphase, and in response to DNA damage throughout the cell cycle.

Pseudosynapsis and decreased stringency of meiotic repair pathway choice on the hemizygous sex chromosome of Caenorhabditis elegans males.

During meiosis, accurate chromosome segregation relies on homology to mediate chromosome pairing, synapsis, and crossover recombination. Crossovers are dependent upon formation and repair of double-strand breaks (DSBs) by homologous recombination (HR). In males of many species, sex chromosomes are largely hemizygous, yet DSBs are induced along nonhomologous regions. Here we analyzed the genetic requirements for meiotic DSB repair on the completely hemizygous X chromosome of Caenorhabditis elegans males. Our data reveal that the kinetics of DSB formation, chromosome pairing, and synapsis are tightly linked in the male germ line. Moreover, DSB induction on the X is concomitant with a brief period of pseudosynapsis that may allow X sister chromatids to masquerade as homologs. Consistent with this, neither meiotic kleisins nor the SMC-5/6 complex are essential for DSB repair on the X. Furthermore, early processing of X DSBs is dependent on the CtIP/Sae2 homolog COM-1, suggesting that as with paired chromosomes, HR is the preferred pathway. In contrast, the X chromosome is refractory to feedback mechanisms that ensure crossover formation on autosomes. Surprisingly, neither RAD-54 nor BRC-2 are essential for DSB repair on the X, suggesting that unlike autosomes, the X is competent for repair in the absence of HR. When both RAD-54 and the structure-specific nuclease XPF-1 are abrogated, X DSBs persist, suggesting that single-strand annealing is engaged in the absence of HR. Our findings indicate that alteration in sister chromatid interactions and flexibility in DSB repair pathway choice accommodate hemizygosity on sex chromosomes.