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Legume GRAS (GAI, RGA, SCR)-type transcription factors MODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

Key messageFor genomic selection in clonally propagated crops with diploid (-like) meiotic behavior to be effective, crossing parents should be selected based on genomic predicted cross-performance unless dominance is negligible.For genomic selection (GS) in clonal breeding programs to be effective, parents should be selected based on genomic predicted cross-performance unless dominance is negligible. Genomic prediction of cross-performance enables efficient exploitation of the additive and dominance value simultaneously. Here, we compared different GS strategies for clonally propagated crops with diploid (-like) meiotic behavior, using strawberry as an example. We used stochastic simulation to evaluate six combinations of three breeding programs and two parent selection methods. The three breeding programs included (1) a breeding program that introduced GS in the first clonal stage, and (2) two variations of a two-part breeding program with one and three crossing cycles per year, respectively. The two parent selection methods were (1) parent selection based on genomic estimated breeding values (GEBVs) and (2) parent selection based on genomic predicted cross-performance (GPCP). Selection of parents based on GPCP produced faster genetic gain than selection of parents based on GEBVs because it reduced inbreeding when the dominance degree increased. The two-part breeding programs with one and three crossing cycles per year using GPCP always produced the most genetic gain unless dominance was negligible. We conclude that (1) in clonal breeding programs with GS, parents should be selected based on GPCP, and (2) a two-part breeding program with parent selection based on GPCP to rapidly drive population improvement has great potential to improve breeding clonally propagated crops.

Fixed nitrogen is one of the most limiting factors for plant growth. The major biological source of fixed nitrogen in ecosystems is provided by nitrogen-fixing bacteria that are able to break the triple bond of N2 molecules and convert it into ammonium via an enzymatic (nitrogenase) activity (Kouchi et al., 2010; Zehr et al., 2003). One of the most important nitrogen-fixing systems is the rhizobium root nodule symbiosis. In this interaction phylogenetically diverse nitrogen fixing rhizobium bacteria colonize nodules on the root of their host plants and find the appropriate conditions to provide fixed nitrogen in exchange for carbohydrates (Kouchi et al., 2010). This efficient endosymbiosis is restricted to two taxonomic lineages: the legume family (Fabaceae) and the genus Parasponia in the Cannabis family (Cannabaceae) (Soltis et al., 1999; Soltis et al., 1995). The ability of these species to achieve a rhizobium endosymbiosis guarantees them a reliable source of nitrogen. In this Thesis I have studied the legume-rhizobium symbiosis, starting from the idea that part of pre-existing signalling pathways have been coopted during evolution of this mutualistic interaction. It is well known that parts of the signalling pathway that is essential for the more ancestral mycorrhizal symbiosis are recruited to support the rhizobium symbiosis (Kouchi et al., 2010; Ivanov et al., 2012). In line with this, it is hypothesized that also other ancestral pathways are recruited during evolution of nodulation. For example, the debate on whether nodule and lateral root formation share parts of their developmental programs has been going on for long (Nutman, 1948; Hirsch et al., 1997; Mathesius et al., 2000; Wopereis et al., 2000; Gonzalez-Rizzo et al., 2006). In Chapter 2 we studied lateral root primordium formation in the model legumes Medicago truncatula (Medicago) and Lotus japonicus (Lotus). Although it is commonly believed that exclusively pericycle cells give rise to the lateral root primordium, similar as seen in Arabidopsis thaliana (Arabidopsis) (Oldroyd et al., 2011; Hirsch et al., 1997), we provide morphological evidence that in the studied legume species this is not the case. In both, Lotus and Medicago, also root cortical cell divisions occur during lateral root formation. Furthermore, we found a striking correlation in the cell layers that are recruited during lateral root and nodule primordium formation. This supports the hypothesis that at least parts of the lateral root developmental program have been recruited during evolution of symbiotic root nodules. Gene duplications -of which a whole genome duplication (WGD) is the most dramatic variant- are known as important driving forces in evolution of new traits. 56 to 65 million years ago an ancestral legume species within the Papilionoidae subfamily (Papilionoids) experienced a WGD event and subsequently gave rise to several major phylogenetic crowns (Lavin et al., 2005). Three of these major lineages are represented by Medicago, Lotus and soybean (Glycine max) (Fawcett et al., 2009; Cannon et al., 2010; Young et al., 2011). I hypothesize that among the orthologous gene pairs maintained in these 3 species are genes that are essential for nodulation. Such genes are yet unidentified by forward genetic screens, because of their (partial) redundancy. I adopted a phylogenetic strategy to identify new candidate genes involved in the legume-Rhizobium symbiosis (Chapter 3, 4 & 5). In a targeted approach, we focussed on the cytokinin phosphorelay pathway, since cytokinin is well known to be involved during nodule organogenesis. This resulted in the identification of one gene pair encoding type-A Response Regulators (RRs). Both these genes, named MtRR9 and MtRR11 in Medicago, are rapidly activated upon rhizobial signalling, whereas all other type-A RR genes are not. Constitutive expression of these type-A RRs is sufficient to trigger cortical cell divisions, suggesting a positive regulatory role for these proteins in root nodule formation. Yet the illustrated role for MtRR9 and MtRR11 in rhizobial symbiosis provides a proof of principle of this method to identify gene pairs involved in legume specific characters (Chapter 3). An unbiased search for paralogous gene pairs revealed two conserved gene duplications in the NADPH oxidases gene family (Young et al., 2011; De Mita & Geurts, unpublished). NADPH oxidases are reactive oxygen species (ROS) producing enzymes. Based on expression pattern it has been speculated that these enzymes are involved in legume-Rhizobium symbiosis (Marino et al., 2010; Chapter 4). So far, such hypothesis was not supported by experimental data. We identified two sets of duplicated genes that have been maintained after the Papilionoid specific WGD (I. MtRBOHA and MtRBOHG and II. MtRBOHE, MtRBOHB, MtRBOHC and MtRBOHD) and aimed to provide support for a symbiotic function for (some of) these genes. In Chapter 4, we show that MtRBOHA and MtRBOHG are redundant, yet essential during symbiosis. In nodules both proteins are associated to rhizobial infection threads at putative sides of bacterial release. MtRBOHA and MtRBOHG are essential during, or just after rhizobium release from infection threads, and their activity sustains the life span of infected cells. Although both genes seem to have redundant functions in symbiosis, they are maintained after the WGD in 3 different legume species. This suggests that there is positive selection to maintain both copies. Though it remains unclear whether this is due to sub- and/or neo-functionalization or due to gene dosage effects (Chapter 4 & 6). The second paralogous gene pair encoding NADPH oxidases underwent additional duplication resulting in 4 genes; MtRBOHE, MtRBOHB, MtRBOHC and MtRBOHD. These genes are phylogenetically positioned in the same orthology group as Arabidopsis AtRHD2. AtRHD2 is a key regulator of root hair tip growth. In Chapter 5 we show that MtRBOHC expression is quickly upregulated upon Rhizobium induced signalling. We postulate that the root hair tip-growth machinery is conserved among species, and that this mechanism is co-opted to support rhizobium root hair based infection. To test this hypothesis MtrbohC knockout mutant was analysed. This mutant did not display an obvious phenotype; symbiotic nor non-symbiotic. This indicates that MtRBOHC is most probably redundant in function.

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

For genomic selection in clonal breeding programs to be effective, crossing parents should be selected based on genomic predicted cross performance unless dominance is negligible. Genomic prediction of cross performance enables a balanced exploitation of the additive and dominance value simultaneously. Here, we compared different strategies for the implementation of genomic selection in clonal plant breeding programs. We used stochastic simulations to evaluate six combinations of three breeding programs and two parent selection methods. The three breeding programs included i) a breeding program that introduced genomic selection in the first clonal testing stage, and ii) two variations of a two-part breeding program with one and three crossing cycles per year, respectively. The two parent selection methods were i) selection of parents based on genomic estimated breeding values, and ii) selection of parents based on genomic predicted cross performance. Selection of parents based on genomic predicted cross performance produced faster genetic gain than selection of parents based on genomic estimated breeding values because it substantially reduced inbreeding when the dominance degree increased. The two-part breeding programs with one and three crossing cycles per year using genomic prediction of cross performance always produced the most genetic gain unless dominance was negligible. We conclude that i) in clonal breeding programs with genomic selection, parents should be selected based on genomic predicted cross performance, and ii) a two-part breeding program with parent selection based on genomic predicted cross performance to rapidly drive population improvement has great potential to improve breeding clonally propagated crops. Competing Interest Statement The authors have declared no competing interest.

ObjectivesTo evaluate the role of the ECG in the differential diagnosis between Anderson-Fabry disease (AFD) and hypertrophic cardiomyopathy (HCM).MethodsIn this multicentre retrospective study, 111 AFD patients with left ventricular hypertrophy were compared with 111 patients with HCM, matched for sex, age and maximal wall thickness by propensity score. Independent ECG predictors of AFD were identified by multivariate analysis, and a multiparametric ECG score-based algorithm for differential diagnosis was developed.ResultsShort PR interval, prolonged QRS duration, right bundle branch block (RBBB), R in augmented vector left (aVL) ≥1.1 mV and inferior ST depression independently predicted AFD diagnosis. A point-by-point ECG score was then derived with the following diagnostic performances: c-statistic 0.80 (95% CI 0.74 to 0.86) for discrimination, the Hosmel-Lemeshow χ2 6.14 (p=0.189) for calibration, sensitivity 69%, specificity 84%, positive predictive value 82% and negative predictive value 72%. After bootstrap resampling, the mean optimism was 0.025, and the internal validated c-statistic for the score was 0.78.ConclusionsStandard ECG can help to differentiate AFD from HCM while investigating unexplained left ventricular hypertrophy. Short PR interval, prolonged QRS duration, RBBB, R in aVL ≥1.1 mV and inferior ST depression independently predicted AFD. Their systematic evaluation and the integration in a multiparametric ECG score can support AFD diagnosis.

•The polyphenol content of the diet was summarized in a dietary index called the polyphenol antioxidant content score.•The polyphenol content of the diet was associated with reduced mortality risk.•The observed effects may be explained by antiinflammatory mechanisms. The effect of the polyphenol content of the human diet on mortality risk is not yet fully understood. The aim of this study was to evaluate the association of a polyphenol-rich diet with mortality rate and a possible mediation effect by inflammation, in what we believe to be a novel, holistic approach. We analyzed 21 302 participants (10 980 women and 10 322 men, aged ≥35 y) from the Moli-sani cohort. The participants were followed up for a median of 8.3 y. The European Prospective Investigation into Cancer and Nutrition food frequency questionnaire (FFQ) was used for dietary assessment. Flavonol, flavone, flavanone, flavanol, anthocyanin, isoflavone, and lignan intakes were calculated using European Food Information Resource—Bioactive Substances in Food Information Systems and the polyphenol antioxidant content (PAC)-score was constructed to assess the total content of these nutrients in the diet. Participants included in the highest quintile of intake of various polyphenol classes and subclasses presented a significant lower all-cause mortality risk compared with those in the lowest group of consumption (hazard ratio [HR] < 1; P < 0.05). Cox regression analyses adjusted for potential confounders indicated that participants in higher quintiles of PAC-score had lower all-cause mortality risk (HR <1; P < 0.05). When cause-specific mortality rates were considered, similar effects were observed for cardiocerebrovascular and cancer mortality (HR <1; P < 0.05). The polyphenol content of the diet was associated with reduced mortality risk in a Mediterranean population, possibly through an antiinflammatory mechanism.

Background Disease-vector mosquito monitoring is an essential prerequisite to optimize control interventions and evidence-based risk predictions. However, conventional entomological monitoring methods are labor- and time-consuming and do not allow high temporal/spatial resolution. In 2022, a novel system coupling an optical sensor with machine learning technologies (VECTRACK) proved effective in counting and identifying Aedes albopictus and Culex pipiens adult females and males. Here, we carried out the first extensive field evaluation of the VECTRACK system to assess: (i) whether the catching capacity of a commercial BG-Mosquitaire trap (BGM) for adult mosquito equipped with VECTRACK (BGM + VECT) was affected by the sensor; (ii) the accuracy of the VECTRACK algorithm in correctly classifying the target mosquito species genus and sex; (iii) Ae. albopictus capture rate of BGM with or without VECTRACK. Methods The same experimental design was implemented in four areas in northern (Bergamo and Padua districts), central (Rome) and southern (Procida Island, Naples) Italy. In each area, three types of traps—one BGM, one BGM + VECT and the combination of four sticky traps (STs)—were rotated each 48 h in three different sites. Each sampling scheme was replicated three times/area. Collected mosquitoes were counted and identified by both the VECTRACK algorithm and operator-mediated morphological examination. The performance of the VECTRACK system was assessed by generalized linear mixed and linear regression models. Aedes albopictus capture rates of BGMs were calculated based on the known capture rate of ST. Results A total of 3829 mosquitoes (90.2% Ae. albopictus ) were captured in 18 collection-days/trap/site. BGM and BGM + VECT showed a similar performance in collecting target mosquitoes. Results show high correlation between visual and automatic identification methods (Spearman Ae. albopictus : females = 0.97; males = 0.89; P  < 0.0001) and low count errors. Moreover, the results allowed quantifying the heterogeneous effectiveness associated with different trap types in collecting Ae. albopictus and predicting estimates of its absolute density. Conclusions Obtained results strongly support the VECTRACK system as a powerful tool for mosquito monitoring and research, and its applicability over a range of ecological conditions, accounting for its high potential for continuous monitoring with minimal human effort. Graphical Abstract

•The Dietary Inflammatory Index measures the potential for dietary inflammation.•This study assessed the association between the index and low-grade inflammation.•To our knowledge, this is the first study to examine this association in an Italian population.•Results showed that proinflammatory diet was associated with higher level of low-grade inflammation. The association between diet and inflammation is well documented. Yet, no evidence exists on the relationship between the inflammatory potential of the diet and low-grade inflammation (LGI) as measured by a composite score of plasma and cellular biomarkers. The aim of this study was to assess the association between the Dietary Inflammatory Index (DII®) and LGI in a large population-based cohort. Cross-sectional analyses were conducted on data from 20 823 adults (age ≥35 y; 48% male) without acute inflammation, who were recruited within the general population of the Moli-sani study from 2005 to 2010. LGI was measured by using a composite score (INFLA-score) including platelet and leukocyte counts, the granulocyte to lymphocyte ratio, and C-reactive protein. DII scores were computed based on dietary intake assessed by the EPIC food frequency questionnaire. Multivariable linear regression models were fit to produce adjusted regression coefficients and 95% confidence intervals (CIs). Higher DII scores were associated with increased LGI (β = 0.131; 95% CI, 0.089–0.174 for the highest versus lowest quintile of DII) after adjusting for age, sex, lifestyle, prevalence of chronic diseases, and health conditions. A higher DII score also was positively associated with each single biomarker of inflammation included in the INFLA-score, unhealthy behaviors (smoking, sedentary lifestyle), and insulin. Higher DII scores, indicating greater inflammatory potential of the diet, were directly associated with LGI, as measured by a composite score of plasma and cellular biomarkers of inflammation. These findings are consistent with the contributing role of diet-mediated inflammation in increasing risk for inflammation-related chronic diseases.