Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions

Large deletions encompassing the NF1 gene and its flanking regions belong to the group of genomic disorders caused by copy number changes that are mediated by the local genomic architecture. Although nonallelic homologous recombination (NAHR) is known to be a major mutational mechanism underlying such genomic copy number changes, the sequence determinants of NAHR location and frequency are still poorly understood since few high-resolution mapping studies of NAHR hotspots have been performed to date. Here, we have characterized two NAHR hotspots, PRS1 and PRS2, separated by 20 kb and located within the low-copy repeats NF1-REPa and NF1-REPc, which flank the human NF1 gene region. High-resolution mapping of the crossover sites identified in 78 type 1 NF1 deletions mediated by NAHR indicated that PRS2 is a much stronger NAHR hotspot than PRS1 since 80% of these deletions exhibited crossovers within PRS2, whereas 20% had crossovers within PRS1. The identification of the most common strand exchange regions of these 78 deletions served to demarcate the cores of the PRS1 and PRS2 hotspots encompassing 1026 and 1976 bp, respectively. Several sequence features were identified that may influence hotspot intensity and direct the positional preference of NAHR to the hotspot cores. These features include regions of perfect sequence identity encompassing 700 bp at the hotspot core, the presence of PRDM9 binding sites perfectly matching the consensus motif for the most common PRDM9 variant, specific pre-existing patterns of histone modification and open chromatin conformations that are likely to facilitate PRDM9 binding

Consideration of the haplotype diversity at nonallelic homologous recombination hotspots improves the precision of rearrangement breakpoint identification

Precise characterization of nonallelic homologous recombination (NAHR) breakpoints is key to identifying those features that influence NAHR frequency. Until now, analysis of NAHR-mediated rearrangements has generally been performed by comparison of the breakpoint-spanning sequences with the human genome reference sequence. We show here that the haplotype diversity of NAHR hotspots may interfere with breakpoint-mapping. We studied the transmitting parents of individuals with germline type-1 NF1 deletions mediated by NAHR within the paralogous recombination site 1 (PRS1) or paralogous recombination site 2 (PRS2) hotspots. Several parental wild-type PRS1 and PRS2 haplotypes were identified that exhibited considerable sequence differences with respect to the reference sequence, which also affected the number of predicted PRDM9-binding sites. Sequence comparisons between the parental wild-type PRS1 or PRS2 haplotypes and the deletion breakpoint-spanning sequences from the patients (method #2) turned out to be an accurate means to assign NF1 deletion breakpoints and proved superior to crude reference sequence comparisons that neglect to consider haplotype diversity (method #1). The mean length of the deletion breakpoint regions assigned by method #2 was 269-bp in contrast to 502-bp by method #1. Our findings imply that paralog-specific haplotype diversity of NAHR hotspots (such as PRS2) and population-specific haplotype diversity must be taken into account in order to accurately ascertain NAHR-mediated rearrangement breakpoints

The Ustilago maydis Effector Pep1 Suppresses Plant Immunity by Inhibition of Host Peroxidase Activity

The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1) as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H2O2 strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction

Charakterisierung der NAHR-Hotspots PRS1 und PRS2, die NF1-Mikrodeletionen vermitteln

Meine Untersuchungen umfassten 78 Patienten mit Typ 1 NF1 Deletionen, welche eine Größe von 1,4 Mb haben und in der Keimbahn eines gesunden Elters entstanden sind (Keimbahn-Deletionen). Typ 1 NF1 Deletionen sind selten und treten mit einer geschätzten Frequenz von 1:60.000 auf. Ursache dieser Deletionen ist eine nicht allelische homologe Rekombination (NAHR) zwischen den beiden paralogen Low-Copy-Repeats NF1-REPa und NF1-REPc. NAHR ist der Mutagenese-Mechanismus, der für die Entstehung von rekurrenten krankheitsassoziierten Deletionen oder Duplikationen im menschlichen Genom verantwortlich ist. Das Ziel meiner Arbeit war eine genaue Kartierung der Deletionsbruchpunkte innerhalb zweier Hotspots für NAHR, den paralogen Rekombinationsstellen 1 und 2 (PRS1 und PRS2). Anhand dieser Kartierung sollten Eigenschaften der betroffenen Bruchpunktbereiche (Strangaustauschregionen, SERs) untersucht werden, die Einfluss auf das Auftreten und die Häufigkeit der vorliegenden NAHR-Ereignisse ausüben könnten. Durch meine Analyse konnte ich die Bruchpunkte der NF1 Deletionen auf SERs von 149 bp bis 1,1 kb eingrenzen. Ein Großteil der Bruchpunkte akkumulierte in Kernbereichen innerhalb der Hotspots. Der Kernbereich in PRS1 von 1 kb enthielt 71 % der in diesem Hotspot lokalisierten Bruchpunkte und der Kernbereich in PRS2 von circa 2 kb enthielt 80 % der Bruchpunkte der PRS2-vermittelten Deletionen. Unter den 78 NF1 Deletionen wiesen 60 (76,5 %) den Bruchpunkt in PRS2 auf, während 18 Deletionen (19,1 %) SERs in PRS1 hatten. Somit ist PRS2 im Vergleich zu PRS1 ein deutlich aktiverer Hotspot mit einer höheren NAHR-Häufigkeit. Bei der genaueren Analyse der beiden Hotspots konnte ich feststellen, dass sich PRS1 und PRS2 hinsichtlich der Anzahl an möglichen Bindungsstellen für das in die Rekombination involvierte Protein PRDM9 unterscheiden. Während in PRS2 vier solcher Bindungsstellen auftreten, liegt in PRS1 nur eine Bindungsstelle, die zudem außerhalb des Kernbereiches lokalisiert ist. Die höhere Anzahl an PRDM9-Bindungsstellen in PRS2 im Vergleich zu PRS1 könnte mit der erhöhten NAHR-Aktivität in PRS2 zusammenhängen. Hohe Sequenzhomologie ist eine Vorrausetzung für NAHR und ausgedehntere Bereiche von Sequenzidentität zwischen paralogen Sequenzen könnten zu einer erhöhten NAHR Rate führen. Hierfür spricht meine Beobachtung, dass nur in PRS2, nicht aber in PRS1 oder flankierenden paralogen Bereichen, eine 700 bp Region perfekter Sequenzhomologie zwischen den beiden NF1-REPs auftritt. Diese Region könnte zu der erhöhten NAHR-Aktivität in PRS2 beitragen. Beide NAHR Hotspots, PRS1 und PRS2, unterscheiden sich von den jeweils flankierenden Sequenzen innerhalb der homologen Bereiche von NF1-REPa und NF1-REPc durch eine offene Chromatinstruktur, die der aktiver Promotoren entspricht. Auch das Muster an Histonmodifikationen ist in diesen Hotspot-Bereichen verändert, im Vergleich zu den flankierenden Regionen, was ebenfalls die NAHR Aktivität in PRS1 und PRS2 begünstigen könnte. Um die SERs bei den Patienten mit NF1 Deletionen genau zu bestimmen, habe ich auch die parentalen Haplotypen der PRS1- und PRS2-Regionen sequenziert. Es fiel auf, dass ein überwiegender Teil der PRS2-Haplotypen beider NF1-REPs nicht vollständig mit der jeweiligen Referenzsequenz übereinstimmte. Es zeigte sich eine Sequenzdiversität, die jedoch bei den verschiedenen Haplotypen unterschiedlich hoch war. Auch zwischen den beiden NF1 REPs waren Unterschiede hinsichtlich der Diversität auffällig. So zeigte sich im Fall von NF1 REPa im Gegensatz zu NF1 REPc eine Gruppe von Haplotypen mit einer geringeren Diversität, also nur wenigen Unterschieden zur Referenzsequenz. Diese PRS2 Haplotypen aus NF1-REPa mit niedriger Diversität waren mit einem Anteil von 58 % relativ häufig, wohingegen solche mit hoher Sequenzdiversität vergleichsweise seltener waren (26 %). Interessanterweise waren Unterschiede bei den Haplotypen verschiedener Sequenzdiversität hinsichtlich der Anzahl der möglichen PRDM9-Bindungsstellen festzustellen. Diese Unterschiede könnten die Häufigkeit der Rekombination beeinflussen. Jedoch konnte ich keinen Einfluss der Höhe der Sequenzdiversität auf die Häufigkeit der NAHR mit Crossover in PRS2 feststellen. Meine Analysen sprechen jedoch dafür, dass die Rate der nicht allelischen homologen Rekombination ohne Crossover (NAHGC) von der unterschiedlichen Sequenzdiversität beeinflusst wird. Die Teilung der PRS2 Haplotypen aus NF1 REPa in zwei Gruppen mit entweder hoher oder niedriger Sequenzdiversität wurde bei den Eltern der Patienten europäischer Abstammung beobachtet. Bei Individuen afrikanischer Abstammung konnte dagegen kein erhöhter Anteil an NF1 REPa Haplotypen mit niedriger Diversität festgestellt werden. Meine Analysen zeigen also deutliche populationsspezifische Unterschiede der Sequenzdiversität im Bereich des PRS2 Hotspots in NF1-REPa. Die hohe Sequenzdiversität mancher PRS2-Haplotypen aus NF1-REPa, die insbesondere bei Afrikanern häufig auftritt, ist demnach anzestral und sehr wahrscheinlich durch NAHGC zwischen den NF1-REPs entstanden, wofür auch der hohen Anteil an shared SNPs (single nucleotide polymorphisms) zwischen NF1-REPa und NF1-REPc spricht. Trotz der hohen Anzahl an shared SNPs ist die Menge an paralogen Sequenzvarianten (PSVs) im Bereich der NAHR Hotspots PRS1 und PRS2 ausreichend, um die Typ 1 Deletionsbruchpunkte genau zu kartieren, was meine Analysen belegen. Damit eignen sich Typ 1 NF1 Deletionen als ein Modellsystem, um die Prozesse zu analysieren, welche die NAHR Hotspots ebenso wie die Häufigkeit der NAHR beeinflussen

<i>In vivo</i> interaction of Pep1 with POX12.

<p>Confocal images in (A) and (B) show <i>N. benthamiana</i> epidermal cells expressing BiFC constructs. (<b>A</b>) A plant cell co-expressing pSPYCE_C and pSPYNE_Pep1. Blue and red channels show apoplastic co-localization of the respective signals. No complementation of fluorescence is observed in the YFP channel. (<b>B</b>) A cell co-expressing pSPYCE_POX12 and pSPYNE_Pep1. Both signals co-localize in the apoplast. The YFP channel exhibits YFP fluorescence with the same localization pattern indicating restoration of the YFP complex due to direct interaction of POX12 and Pep1. Bars: 25 µm. (<b>C</b>) Yeast-Two-Hybrid experiment confirming interaction of Pep1 and POX12. Mutation of the putative active site of POX12 (POX12_m) did not abolish interaction with Pep1.</p

Inhibition of the elicitor triggered oxidative burst in maize leaves.

<p>(<b>A</b>) Luminol based readout to determine H₂O₂ production in maize leaf discs. The oxidative burst was elicited by the addition of chitosan (2.5 mg/ml) one minute after starting the measurement. Concentrations of recombinant Pep1, Pep1_IA and GFP proteins: 10 µM. (<b>B</b>) Quantification of elicitor triggered H₂O₂ production in maize leaf discs. The bars represent the integrated signal intensity of the average of 6 independent samples over the first 5 min after elicitation. (<b>C</b>) Quantification of chitosan induced H₂O₂ production in maize leaf discs based on xylenol orange staining. The peroxidase inhibitor SHAM (2 mM), NADPH-oxidase inhibitor DPI (5 µM) as well recombinant Pep1 protein (10 µM) cause a significant reduction of H₂O₂. Heat inactivated Pep1 (Pep1_IA) does not influence the elicitor triggered oxidative burst. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Scavenging of reactive oxygen species suppresses maize penetration resistance to the <i>Δpep1</i> mutant.

<p>(<b>A</b>) Aniline blue staining of SG200Δpep1RFP attempting to penetrate maize epidermis cell, 24 hpi. A papilla is formed at the point of penetration (arrow). (<b>B</b>) SG200Δpep1RFP penetrating the maize epidermis of plants treated with 5 µM ascorbate. Arrows mark penetration sites. The invading hyphae succeed in cell to cell penetrations (arrow heads); in some cases, proliferation of the hypha could be observed (chevron). (<b>C</b>) SG200Δpep1RFP on maize leaves expressing PIN1-YFP. Enhanced autofluorescence of penetrated cells as well as cells surrounding the penetration event indicate a HR reaction of the plant. (<b>D</b>) SG200Δpep1RFP is able to penetrate PIN1-YFP expressing maize leaves after the treatment with 5 µM ascorbate without eliciting enhanced autofluorescence. Bars: 20 µm. (<b>E</b>) Quantification of the length of intracellularly growing hyphae of SG200Δpep1 in maize leaves treated with 5 µM ascorbate compared to mock treated leaves. The addition of ascorbate leads to an average 6-fold increase of hyphal length. (<b>F</b>) Quantification of maize epidermal cells expressing visual signs of HR per penetration attempt by SG200Δpep1. The ascorbate treated plants show a ∼45% decrease in HR symptoms compared to the mock treated plants. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Regulation of JA and SA associated maize marker genes in response to <i>U. maydis</i> wild type versus SG200Δpep1 infections.

<p>Expression levels of SA/JA marker genes were determined by quantitative real-time PCR. The expression values represent three biological replicates and are shown relative to <i>GAPDH</i> expression in each sample. Leaf samples of mock, SG200 or SG200Δpep1 infected plants were taken after 2 dpi. Expression levels in mock infected plants were set to 1 and relative expression of marker genes was calculated for SG200 (light grey bars) and SG200Δpep1 (dark grey bars) samples. (<b>A</b>) Expression of SA marker genes <i>atfp4</i>, <i>pox12</i> and <i>pr1</i> 24 hours after infection with strain SG200Δpep1 or SG200, respectively. (<b>B</b>) Expression of JA marker genes <i>cc9</i> and <i>bbi</i> after infection with strain SG200Δpep1 or SG200, respectively. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Pep1 directly inhibits peroxidase activity.

<p>(<b>A</b>) Measurement of HRP activity using an <i>in vitro</i> DAB assay. Dark coloration indicates peroxidase activity, visualized by the precipitation of DAB. The addition of purified GFP as well as heat inactivated Pep1 (Pep1_IA) does not interfere with HRP activity. Addition of 5 µM native Pep1 results in reduced DAB precipitation, indicating suppressed HRP activity. (<b>B</b>) Quantification of DAB based HRP activity assay. Different concentrations of Pep1 were added to the assay solution at two different relevant pH values. Pep1 exhibits the ability of concentration dependent suppression of HRP activity at pH 6.5 and 7.5. (<b>C</b>) Far Western blot shows physical interaction of HRP with Pep1 (18.5 kDa) but not GFP (29.8 kDa) (upper panel). As a loading control, a separate gel was equally loaded and stained with coomassie blue (lower panel). (<b>D</b>) Peroxidase activity of maize apoplastic fluid was determined in a quantitative DAB assay. Recombinant native Pep1 and heat inactivated Pep1 (Pep1_IA) were added to the reaction in respective concentrations. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p

Silencing of <i>pox12</i> suppresses maize penetration resistance to the <i>Δpep1</i> mutant.

<p>(<b>A</b>) (left panel): Aniline blue staining of control plants (BMV-YFPsi) show formation of papillae at points of SG200Δpep1 penetration attempts. SG200Δpep1 is arrested directly upon penetration. (right panel): <i>pox12</i>-silenced (BMV-POX12si) maize plants infected with SG200Δpep1. Strain SG200Δpep1 successfully penetrates epidermal cells (arrows), shows cell to cell penetrations (arrow heads) and reaches the mesophyll layer (M, chevron) without eliciting visible plant defense responses. Bars: 10 µm (<b>B</b>) Quantification of intracellular hyphae length of <i>U. maydis</i> SG200Δpep1 on <i>pox12</i>-silencing plants compared to control plants. Silencing of <i>pox12</i> led to a significant, ∼10-fold increase in length of intracellular SG200Δpep1 hyphae. (<b>C</b>) <i>pox12</i> expression was quantified by quantitative real-time PCR using leaf samples of 8 independent <i>pox12</i>-silenced plants (BMV/POX12si) and 7 control plants (BMV/YFPsi) 48 h after <i>U. maydis</i> SG200Δpep1 infection (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002684#s4" target="_blank">methods</a> section). Relative expression of <i>pox12</i> in BMV/YFPsi control plants was averaged and set to 1. Data represent three biological replicates. P values have been calculated by an unpaired <i>t</i> test. Error bars show SEM. * P≤0.05.</p