Physical immune escape: Weakened mechanical communication leads to escape of metastatic colorectal carcinoma cells from macrophages.

The significance of biochemical cues in the tumor immune microenvironment in affecting cancer metastasis is well established, but the role of physical factors in the microenvironment remains largely unexplored. In this article, we investigated how the mechanical interaction between cancer cells and immune cells, mediated by extracellular matrix (ECM), influences immune escape of cancer cells. We focus on the mechanical regulation of macrophages' targeting ability on two distinct types of colorectal carcinoma (CRC) cells with different metastatic potentials. Our results show that macrophages can effectively target CRC cells with low metastatic potential, due to the strong contraction exhibited by the cancer cells on the ECM, and that cancer cells with high metastatic potential demonstrated weakened contractions on the ECM and can thus evade macrophage attack to achieve immune escape. Our findings regarding the intricate mechanical interactions between immune cells and cancer cells can serve as a crucial reference for further exploration of cancer immunotherapy strategies.

Transcription factor encoding gene OsC1 regulates leaf sheath color through anthocyanidin metabolism in Oryza rufipogon and Oryza sativa.

Carbohydrates, proteins, lipids, minerals and vitamins are nutrient substances commonly seen in rice grains, but anthocyanidin, with benefit for plant growth and animal health, exists mainly in the common wild rice but hardly in the cultivated rice. To screen the rice germplasm with high intensity of anthocyanidins and identify the variations, we used metabolomics technique and detected significant different accumulation of anthocyanidins in common wild rice (Oryza rufipogon, with purple leaf sheath) and cultivated rice (Oryza sativa, with green leaf sheath). In this study, we identified and characterized a well-known MYB transcription factor, OsC1, through phenotypic (leaf sheath color) and metabolic (metabolite profiling) genome-wide association studies (pGWAS and mGWAS) in 160 common wild rice (O. rufipogon) and 151 cultivated (O. sativa) rice varieties. Transgenic experiments demonstrated that biosynthesis and accumulation of cyanidin-3-Galc, cyanidin 3-O-rutinoside and cyanidin O-syringic acid, as well as purple pigmentation in leaf sheath were regulated by OsC1. A total of 25 sequence variations of OsC1 constructed 16 functional haplotypes (higher accumulation of the three anthocyanidin types within purple leaf sheath) and 9 non-functional haplotypes (less accumulation of anthocyanidins within green leaf sheath). Three haplotypes of OsC1 were newly identified in our germplasm, which have potential values in functional genomics and molecular breeding of rice. Gene-to-metabolite analysis by mGWAS and pGWAS provides a useful and efficient tool for functional gene identification and omics-based crop genetic improvement.

Creation and gene expression analysis of a giant embryo rice mutant with high GABA content.

Gamma-amino butyric acid (GABA) is a natural non-protein amino acid involved in stress, signal transmission, carbon and nitrogen balance, and other physiological processes in plants. In the human body, GABA has the effects of lowering blood pressure, anti-aging, and activating the liver and kidneys. However, there are few studies on the molecular regulation mechanism of genes in the metabolic pathways of GABA during grain development of giant embryo rice with high GABA content. In this study, three glant embryo (ge) mutants of different embryo sizes were obtained by CRISPR/Cas9 knockout, and it was found that GABA, protein, crude fat, and various mineral contents of the ge mutants were significantly increased. RNA-seq and qRT-PCR analysis showed that in the GABA shunt and polyamine degradation pathways, the expression levels of most of the genes encoding enzymes promoting GABA accumulation were significantly upregulated in the ge-1 mutant, whereas, the expression levels of most of the genes encoding enzymes involved GABA degradation were significantly downregulated in the ge-1 mutant. This is most likely responsible for the significant increase in GABA content of the ge mutant. These results help reveal the molecular regulatory network of GABA metabolism in giant embryo rice and provide a theoretical basis for the study of its development mechanisms, which is conducive to the rapid cultivation of GABA-rich rice varieties, promoting human nutrition, and ensuring health. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01353-1.

The genetic basis of grain protein content in rice by genome-wide association analysis.

The grain protein content (GPC) of rice is an important factor that determines its nutritional, cooking, and eating qualities. To date, although a number of genes affecting GPC have been identified in rice, most of them have been cloned using mutants, and only a few genes have been cloned in the natural population. In this study, 135 significant loci were detected in a genome-wide association study (GWAS), many of which could be repeatedly detected across different years and populations. Four minor quantitative trait loci affecting rice GPC at four significant association loci, qPC2.1, qPC7.1, qPC7.2, and qPC1.1, were further identified and validated in near-isogenic line F2 populations (NIL-F2), explaining 9.82, 43.4, 29.2, and 13.6% of the phenotypic variation, respectively. The role of the associated flo5 was evaluated with knockdown mutants, which exhibited both increased grain chalkiness rate and GPC. Three candidate genes in a significant association locus region were analyzed using haplotype and expression profiles. The findings of this study will help elucidate the genetic regulatory network of protein synthesis and accumulation in rice through cloning of GPC genes and provide new insights on dominant alleles for marker-assisted selection in the genetic improvement of rice grain quality. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01347-z.

Improvement of Rice Blast Resistance in TGMS Line HD9802S through Optimized Anther Culture and Molecular Marker-Assisted Selection.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. The early indica rice thermosensitive genic male sterile (TGMS) line HD9802S has the characteristics of stable fertility, reproducibility, a high outcrossing rate, excellent rice quality, and strong combining ability. However, this line exhibits poor blast resistance and is highly susceptible to leaf and neck blasts. In this study, backcross introduction, molecular marker-assisted selection, gene chipping, anther culture, and resistance identification in the field were used to introduce the broad-spectrum blast-resistance gene R6 into HD9802S to improve its rice blast resistance. Six induction media were prepared by varying the content of each component in the culture medium. Murashige and Skoog's medium with 3 mg/L 2,4-dichlorophenoxyacetic acid, 2 mg/L 1-naphthaleneacetic acid, and 1 mg/L kinetin and N6 medium with 800 mg/L casein hydrolysate, 600 mg/L proline, and 500 mg/L glutamine could improve the callus induction rate and have a higher green seedling rate and a lower white seedling rate. Compared to HD9802S, two doubled haploid lines containing R6 with stable fertility showed significantly enhanced resistance to rice blast and no significant difference in spikelet number per panicle, 1000-grain weight, or grain shape. Our findings highlight a rapid and effective method for improving rice blast resistance in TGMS lines.

Bivariate categorical data analysis using normal linear conditional multinomial probability model.

Bivariate multinomial data such as the left and right eyes retinopathy status data are analyzed either by using a joint bivariate probability model or by exploiting certain odds ratio-based association models. However, the joint bivariate probability model yields marginal probabilities, which are complicated functions of marginal and association parameters for both variables, and the odds ratio-based association model treats the odds ratios involved in the joint probabilities as 'working' parameters, which are consequently estimated through certain arbitrary 'working' regression models. Also, this later odds ratio-based model does not provide any easy interpretations of the correlations between two categorical variables. On the basis of pre-specified marginal probabilities, in this paper, we develop a bivariate normal type linear conditional multinomial probability model to understand the correlations between two categorical variables. The parameters involved in the model are consistently estimated using the optimal likelihood and generalized quasi-likelihood approaches. The proposed model and the inferences are illustrated through an intensive simulation study as well as an analysis of the well-known Wisconsin Diabetic Retinopathy status data.

MKK3 Cascade Regulates Seed Dormancy Through a Negative Feedback Loop Modulating ABA Signal in Rice.

BACKGROUND: With the increasing frequency of climatic anomalies, high temperatures and long-term rain often occur during the rice-harvesting period, especially for early rice crops in tropical and subtropical regions. Seed dormancy directly affects the resistance to pre-harvest sprouting (PHS). Therefore, in order to increase rice production, it is critical to enhance seed dormancy and avoid yield losses to PHS. The elucidation and utilization of the seed dormancy regulation mechanism is of great significance to rice production. Preliminary results indicated that the OsMKKK62-OsMKK3-OsMPK7/14 module might regulate ABA sensitivity and then control seed dormancy. The detailed mechanism is still unclear. RESULTS: The overexpression of OsMKK3 resulted in serious PHS. The expression levels of OsMKK3 and OsMPK7 were upregulated by ABA and GA at germination stage. OsMKK3 and OsMPK7 are both located in the nucleus and cytoplasm. The dormancy level of double knockout mutant mkk3/mft2 was lower than that of mkk3, indicating that OsMFT2 functions in the downstream of MKK3 cascade in regulating rice seeds germination. Biochemical results showed that OsMPK7 interacted with multiple core ABA signaling components according to yeast two-hybrid screening and luciferase complementation experiments, suggesting that MKK3 cascade regulates ABA signaling by modulating the core ABA signaling components. Moreover, the ABA response and ABA responsive genes of mpk7/14 were significantly higher than those of wild-type ZH11 when subjected to ABA treatment. CONCLUSION: MKK3 cascade mediates the negative feedback loop of ABA signal through the interaction between OsMPK7 and core ABA signaling components in rice.

OsLSC6 Regulates Leaf Sheath Color and Cold Tolerance in Rice Revealed by Metabolite Genome Wide Association Study.

Plant metabolites including anthocyanins play an important role in the growth of plants, as well as in regulating biotic and abiotic stress responses to the environment. Here we report comprehensive profiling of 3315 metabolites and a further metabolic-based genome-wide association study (mGWAS) based on 292,485 SNPs obtained from 311 rice accessions, including 160 wild and 151 cultivars. We identified hundreds of common variants affecting a large number of secondary metabolites with large effects at high throughput. Finally, we identified a novel gene namely OsLSC6 (Oryza sativa leaf sheath color 6), which encoded a UDP 3-O-glucosyltransferase and involved in the anthocyanin biosynthesis of Cyanidin-3-Galc (sd1825) responsible for leaf sheath color, and resulted in significant different accumulation of sd1825 between wild (purple) and cultivars (green). The results of knockout transgenic experiments showed that OsLSC6 regulated the biosynthesis and accumulation of sd1825, controlled the purple leaf sheath. Our further research revealed that OsLSC6 also confers resistance to cold stress during the seedling stage in rice. And we identified that a SNP in OsLSC6 was responsible for the leaf sheath color and chilling tolerance, supporting the importance of OsLSC6 in plant adaption. Our study could not only demonstrate that OsLSC6 is a vital regulator during anthocyanin biosynthesis and abiotic stress responses, but also provide a powerful complementary tool based on metabolites-to-genes analysis by mGWAS for functional gene identification andpromising candidate in future rice breeding and improvement.

Root microbiota analysis of Oryza rufipogon and Oryza sativa reveals an orientation selection during the domestication process.

The root-associated microbiota has a close relation to the life activities of plants, and its composition is affected by the rhizospheric environment and plant genotypes. Rice (Oryza sativa) was domesticated from the ancestor species Oryza rufipogon. Many important agricultural traits and adversity resistance of rice have changed during a long time of natural domestication and artificial selection. However, the influence of rice genotypes on root microbiota in important agricultural traits remains to be explained. In this study, we performed 16S rRNA and internal transcribed spacer (ITS) gene amplicon sequencing to generate bacterial and fungal community profiles of O. rufipogon and O. sativa, both of which were planted in a farm in Guangzhou and had reached the reproductive stage. We compared their root microbiota in detail by alpha diversity, beta diversity, different species, core microbiota, and correlation analyses. We found that the relative abundance of bacteria was significantly higher in the cultivated rice than in the common wild rice, while the relative abundance of fungi was the opposite. Significant differences in agricultural traits between O. rufipogon and O. sativa showed a high correlation with core microorganisms in the two Oryza species, which only existed in either or had obviously different abundance in both two species, indicating that rice genotype/phenotype had a strong influence on recruiting specific microorganisms. Our study provides a theoretical basis for the in-depth understanding of rice root microbiota and the improvement of rice breeding from the perspective of the interaction between root microorganisms and plants.IMPORTANCEPlant root microorganisms play a vital role not only in plant growth and development but also in responding the biotic and abiotic stresses. Oryza sativa is domesticated from Oryza rufipogon which has many excellent agricultural traits especially containing resistance to biotic and abiotic stresses. To improve the yield and resistance of cultivated rice, it is particularly important to deeply research on differences between O. sativa and O. rufipogon and find beneficial microorganisms to remodel the root microbiome of O. sativa.

Transcriptome and Metabolome Analysis of Rice Cultivar CBB23 after Inoculation by Xanthomonas oryzae pv. oryzae Strains AH28 and PXO99A.

Bacterial leaf blight (BLB), among the most serious diseases in rice production, is caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23, the broadest resistance gene against BLB in rice, is widely used in rice breeding. In this study, the rice variety CBB23 carrying the Xa23 resistance gene was inoculated with AH28 and PXO99A to identify differentially expressed genes (DEGs) associated with the resistance. Transcriptome sequencing of the infected leaves showed 7997 DEGs between the two strains at different time points, most of which were up-regulated, including cloned rice anti-blight, peroxidase, pathology-related, protein kinase, glucosidase, and other coding genes, as well as genes related to lignin synthesis, salicylic acid, jasmonic acid, and secondary metabolites. Additionally, the DEGs included 40 cloned, five NBS-LRR, nine SWEET family, and seven phenylalanine aminolyase genes, and 431 transcription factors were differentially expressed, the majority of which belonged to the WRKY, NAC, AP2/ERF, bHLH, and MYB families. Metabolomics analysis showed that a large amount of alkaloid and terpenoid metabolite content decreased significantly after inoculation with AH28 compared with inoculation with PXO99A, while the content of amino acids and their derivatives significantly increased. This study is helpful in further discovering the pathogenic mechanism of AH28 and PXO99A in CBB23 rice and provides a theoretical basis for cloning and molecular mechanism research related to BLB resistance in rice.

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide, jasmonic acid, and phytoalexin metabolic pathways.

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H2 O2 ) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H2 O2 in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.

Hyperspectral Imaging during Normothermic Machine Perfusion-A Functional Classification of Ex Vivo Kidneys Based on Convolutional Neural Networks.

Facing an ongoing organ shortage in transplant medicine, strategies to increase the use of organs from marginal donors by objective organ assessment are being fostered. In this context, normothermic machine perfusion provides a platform for ex vivo organ evaluation during preservation. Consequently, analytical tools are emerging to determine organ quality. In this study, hyperspectral imaging (HSI) in the wavelength range of 550-995 nm was applied. Classification of 26 kidneys based on HSI was established using KidneyResNet, a convolutional neural network (CNN) based on the ResNet-18 architecture, to predict inulin clearance behavior. HSI preprocessing steps were implemented, including automated region of interest (ROI) selection, before executing the KidneyResNet algorithm. Training parameters and augmentation methods were investigated concerning their influence on the prediction. When classifying individual ROIs, the optimized KidneyResNet model achieved 84% and 62% accuracy in the validation and test set, respectively. With a majority decision on all ROIs of a kidney, the accuracy increased to 96% (validation set) and 100% (test set). These results demonstrate the feasibility of HSI in combination with KidneyResNet for non-invasive prediction of ex vivo kidney function. This knowledge of preoperative renal quality may support the organ acceptance decision.

Genetic diversity of wild rice accessions (Oryza rufipogon Griff.) in Guangdong and Hainan Provinces, China, and construction of a wild rice core collection.

Oryza rufipogon Griff. is a valuable germplasm resource for rice genetic improvement. However, natural habitat loss has led to the erosion of the genetic diversity of wild rice populations. Genetic diversity analysis of O. rufipogon accessions and development of the core collection are crucial for conserving natural genetic diversity and providing novel traits for rice breeding. In the present study, we developed 1,592 SNPs by multiplex PCR and next-generation sequencing (NGS) technology and used them to genotype 998 O. rufipogon accessions from 14 agroclimatic zones in Guangdong and Hainan Provinces, China. These SNPs were mapped onto 12 chromosomes, and the average MAF value was 0.128 with a minimum of 0.01 and a maximum of 0.499. The O. rufipogon accessions were classified into ten groups. The mean Nei's diversity index and Shannon-Wiener index (I) were 0.187 and 0.308, respectively, in all populations, indicating that O. rufipogon accessions had rich genetic diversity. There were also differences in the genetic diversity of O. rufipogon resources in the 14 regions. Hainan populations possessed higher levels of genetic diversity, whereas the Guangzhou population had lower levels of genetic diversity than did the other populations. Phylogenetic analysis revealed that the genetic relationship among the distribution sites of O. rufipogon was closely related to geographical location. Based on genetic distance, a core collection of 299 accessions captured more than 99% of the genetic variation in the germplasm. This study provides insights into O. rufipogon conservation, and the constructed core collection provides valuable resources for future research and genomics-assisted breeding of rice.

Genomic and transcriptomic analysis reveal molecular basis of salinity tolerance in a novel strong salt-tolerant rice landrace Changmaogu.

BACKGROUND: Salt stress is an important factor that limits rice yield. We identified a novel, strongly salt tolerant rice landrace called Changmaogu (CMG) collected from a coastal beach of Zhanjiang, Guangdong Province, China. The salt tolerance of CMG was much better than that of the international recognized salt tolerant rice cultivar Pokkali in the germination and seedling stages. RESULTS: To understand the molecular basis of salt tolerance in CMG, we performed BSA-seq for two extreme bulks derived from the cross between CMG and a cultivar sensitive to salt, Zhefu802. Transcriptomic sequencing was conducted for CMG at the germination and young seedling stages. Six candidate regions for salt tolerance were mapped on Chromosome 1 by BSA-seq using the extreme populations. Based on the polymorphisms identified between both parents, we detected 32 genes containing nonsynonymous coding single nucleotide polymorphisms (SNPs) and frameshift mutations in the open reading frame (ORF) regions. With transcriptomic sequencing, we detected a large number of differentially expressed genes (DEGs) at the germination and seedling stages under salt stress. KEGG analysis indicated two of 69 DEGs shared at the germination and seedling stages were significantly enriched in the pathway of carotenoid biosynthesis. Of the 169 overlapping DEGs among three sample points at the seedling stage, 13 and six DEGs were clustered into the pathways of ABA signal transduction and carotenoid biosynthesis, respectively. Of the 32 genes carrying sequence variation, only OsPP2C8 (Os01g0656200) was differentially expressed in the young seedling stage under salt stress and also showed sequence polymorphism in the ORFs between CMG and Zhefu802. CONCLUSION: OsPP2C8 was identified as the target candidate gene for salinity tolerance in the seedling stage. This provides an important genetic resource for the breeding of novel salt tolerant rice cultivars.

Genetic and cytological analysis of a novel type of low temperature-dependent intrasubspecific hybrid weakness in rice.

Hybrid weakness (HW) is an important postzygotic isolation which occurs in both intra- and inter-specific crosses. In this study, we described a novel low temperature-dependent intrasubspecific hybrid weakness in the F1 plants derived from the cross between two indica rice varieties Taifeng A and V1134. HW plants showed growth retardation, reduced panicle number and pale green leaves with chlorotic spots. Cytological assay showed that there were reduced cell numbers, larger intercellular spaces, thicker cell walls, and abnormal development of chloroplast and mitochondria in the mature leaves from HW F1 plants in comparison with that from both of the parental lines. Genetic analysis revealed that HW was controlled by two complementary dominant genes Hw3 from V1134 and Hw4 from Taifeng A. Hw3 was mapped in a 136 kb interval between the markers Indel1118 and Indel1117 on chromosome 11, and Hw4 was mapped in the region of about 15 cM between RM182 and RM505 on chromosome 7, respectively. RT-PCR analysis revealed that only LOC_Os11g44310, encoding a putative calmodulin-binding protein (OsCaMBP), differentially expressed among Taifeng A, V1134 and their HW F1. No recombinant was detected using the markers designed based on the sequence of LOC_Os11g44310 in the BC1F2 (Taifeng A//Taifeng A/V1134) population. Hence, LOC_Os11g44310 was probably the candidate gene of Hw3. Gene amplification suggested that LOC_Os11g44310 was present in V1134 and absent in Taifeng A. BLAST search revealed that LOC_Os11g44310 had one copy in the japonica genomic sequence of Nipponbare, and no homologous sequence in the indica reference sequence of 9311. Our results indicate that Hw3 is a novel gene for inducing hybrid weakness in rice.