The status of the CRISPR/Cas9 research in plant-nematode interactions.

MAIN CONCLUSION: As an important biotic stressor, plant-parasitic nematodes afflict global crop productivity. Deployment of CRISPR/Cas9 system that selectively knock out host susceptibility genes conferred improved nematode tolerance in crop plants. As an important biotic stressor, plant-parasitic nematodes cause a considerable yield decline in crop plants that eventually contributes to a negative impact on global food security. Being obligate plant parasites, the root-knot and cyst nematodes maintain an intricate and sophisticated relationship with their host plants by hijacking the host's physiological and metabolic pathways for their own benefit. Significant progress has been made toward developing RNAi-based transgenic crops that confer nematode resistance. However, the strategy of host-induced gene silencing that targets nematode effectors is likely to fail because the induced silencing of effectors (which interact with plant R genes) may lead to the development of nematode phenotypes that break resistance. Lately, the CRISPR/Cas9-based genome editing system has been deployed to achieve host resistance against bacteria, fungi, and viruses. In these studies, host susceptibility (S) genes were knocked out to achieve resistance via loss of susceptibility. As the S genes are recessively inherited in plants, induced mutations of the S genes are likely to be long-lasting and confer broad-spectrum resistance. A number of S genes contributing to plant susceptibility to nematodes have been identified in Arabidopsis thaliana, rice, tomato, cucumber, and soybean. A few of these S genes were targeted for CRISPR/Cas9-based knockout experiments to improve nematode tolerance in crop plants. Nevertheless, the CRISPR/Cas9 system was mostly utilized to interrogate the molecular basis of plant-nematode interactions rather than direct research toward achieving tolerance in crop plants. The current standalone article summarizes the progress made so far on CRISPR/Cas9 research in plant-nematode interactions.

Marker-assisted enhancement of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in a salt-tolerant rice variety for sustaining rice production of tropical islands.

Introduction: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice, specially in the tropical regions of the world. Developing rice varieties with host resistance against the disease is the most effective and economical solution for managing the disease. Methods: Pyramiding resistance genes (Xa4, xa5, xa13,and Xa21) in popular rice varieties using marker-assisted backcross breeding (MABB) has been demonstrated as a cost-effective and sustainable approach for establishing durable BB resistance. Here, we report our successful efforts in introgressing four resistance genes (Xa4, xa5, xa13, and Xa21) from IRBB60 to CARI Dhan 5, a popular salt-tolerant variety developed from a somaclonal variant of Pokkali rice, through functional MABB. Results and discussion: Both BB and coastal salinity are among the major challenges for rice production in tropical island and coastal ecosystems. Plants with four, three, and two gene pyramids were generated, which displayed high levels of resistance to the BB pathogen at the BC3F2 stage. Under controlled salinity microplot environments, the line 131-2-175-1223 identified with the presence of three gene pyramid (Xa21+xa13+xa5) displayed notable resistance across locations and years as well as exhibited a salinity tolerance comparable to the recurrent parent, CARI Dhan 5. Among two BB gene combinations (Xa21+xa13), two lines, 17-1-69-334 and 46-3-95-659, demonstrated resistance across locations and years, as well as salt tolerance and grain production comparable to CARI Dhan 5. Besides salinity tolerance, five lines, 17-1-69-179, 46-3-95-655, 131-2-190-1197, 131-2-175-1209, and 131-2-175-1239, exhibited complete resistance to BB disease. Following multilocation testing, potential lines have been identified that can serve as a prospective candidate for producing varieties for the tropical Andaman and Nicobar Islands and other coastal locations, which are prone to BB and coastal salinity stresses.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy.

BACKGROUND: Plant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease. RESULTS: Initially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1-3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, 'transgene-free' T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants. CONCLUSION: Our results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

Selection of suitable internal control gene for assaying gene expression in rice through qRT-PCR during sheath blight infection.

qRT-PCR is a globally accepted technique for assaying gene expression in relative terms which compares the difference between critical threshold (CT) values of a gene calculated form two independently isolated RNA samples. Independent RNA isolations, however, include error due to batch effect which must be normalized for error-free calculation of relative gene expression. Hence, CT values of internal control (IC) genes are used for normalization during the calculation of expression fold-change in gene expression analysis. The expression of ICs genes expected to be stable in all the experimental conditions. However, it is almost impossible to find such a gene which do not depict expression fluctuation in response to the changes in experimental conditions. Hence, it is necessary to identify suitable IC gene(s) for any given experimental condition before conducting any particular gene expression study. Here, we examined the suitability of eight candidate IC genes, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic elongation factor-1 (eEF-1α), 25 S rRNA (25 S), 18 S rRNA (18 S), ubiquitin C E2 ligase (UBC), Actin (Act), ubiquitin 5 (UBQ5) and ubiquitin 10 (UBQ10), for assaying gene expression in rice during sheath blight infection. Our analysis suggest that GAPDH might be the IC of choice when expression studies include contrasting genotypes differing in their tolerance to sheath blight pathogen as well as progressive infection time. While if expression analysis have to be performed only in one genotype but under progressive sheath blight infection, UBQ5 might be chosen as IC because of its high expression stability under the proposed experimental setup.

Comparative transcriptome profiling reveals the basis of differential sheath blight disease response in tolerant and susceptible rice genotypes.

Rice sheath blight (ShB) disease, caused by the fungal pathogen Rhizoctonia solani AG1-IA, is one of the devastating diseases and causes severe yield losses all over the world. No completely resistant germplasm is known till now, and as a result, the progress in resistance breeding is unsatisfactory. Basic studies to identify candidate genes, QTLs, and to better understand the host-pathogen interaction are also scanty. In this study, we report the identification of a new ShB-tolerant rice germplasm, CR 1014. Further, we investigated the basis of tolerance by exploring the disease responsive differentially expressed transcriptome and comparing them with that of a susceptible variety, Swarna-Sub1. A total of 815 and 551 genes were found to be differentially regulated in CR 1014 and Swarna-Sub1, respectively, at two different time points. The result shows that the ability to upregulate genes for glycosyl hydrolase, secondary metabolite biosynthesis, cytoskeleton and membrane integrity, the glycolytic pathway, and maintaining photosynthesis make CR 1014 a superior performer in resisting the ShB pathogen. We discuss several putative candidate genes for ShB resistance. The present study, for the first time, revealed the basis of ShB tolerance in the germplasm CR1014 and should prove to be particularly valuable in understanding molecular response to ShB infection. The knowledge could be utilized to devise strategies to manage the disease better.

Preformed aerenchyma determines the differential tolerance response under partial submergence imposed by fresh and saline water flooding in rice.

Plant's response to fresh- and saline-water flooding and the resulting partial submergence, seems different due to the added complexities of element toxicity of salinity. We identified a few rice genotypes which can tolerate combined stresses of partial submergence and salinity during saline water flooding. To gain mechanistic insights, we compared two rice genotypes: Varshadhan (freshwater-flooding tolerant) and Rashpanjor (both fresh- and saline-water flooding tolerant). We found greater ethylene production and increased "respiratory burst oxidase homolog" (RBOH)-mediated reactive oxygen species (ROS) production led to well-developed constitutive aerenchyma formation in Rashpanjor, which makes it preadapted to withstand fresh- and saline-water flooding. On the contrary, an induced aerenchyma formation-dependent tolerance mechanism of Varshadhan worked well for freshwater flooding but failed to provide tolerance to saline-water flooding. Additional salt stress was found to significantly inhibit the induced aerenchyma formation process due to the dampening of ROS signaling by the action of metallothionein in Varshadhan. Besides, inconspicuous changes in ionic regulation processes in these two genotypes under saline-water flooding suggest preadapted constitutive aerenchyma formation plays a more significant role than elemental toxicity per se in tolerating combined stresses encountered during saline water flooding in rice. Overall, our study indicated that well-developed constitutive aerenchyma provide an adaptive advantage during partial submergence due to saline water flooding in rice as the key process of induced aerenchyma formation is hampered in the presence of salinity stress coupled with partial submergence.

Rice with SUB1 QTL possesses greater initial leaf gas film thickness leading to delayed perception of submergence stress.

BACKGROUND AND AIMS: Submergence tolerance in rice is primarily attributed to the action of the SUB1 gene, but other associated traits such as leaf gas film (LGF) thickness, leaf hydrophobicity, porosity and leaf density have been known to aid submergence tolerance in rice. However, association of these traits with SUB1 quantitative trait locus (QTL) has not been demonstrated. In this study, we aim to investigate (1) whether the presence of the SUB1 QTL in the genetic background has any influence on the thickness of the LGF and (ii) whether its removal has any impact on stress perception and submergence tolerance in Sub1 and non-Sub1 rice. METHODS: We examined 12 genotypes (including both Sub1 and non-Sub1 types) for different leaf traits such as initial LGF thickness, leaf hydrophobicity, tissue porosity and leaf density in order to work out the relatioship of these traits to the SUB1 QTL in rice. Furthermore, we investigated the changes in the gene expression profile and different metabolic processes in selected genotypes in the presence and absence of their LGF to study its impact on stress perception and adaptation. KEY RESULTS: The initial thickness of the LGF and hydrophobicity seemed to have a highly positive correlation with the presence of the SUB1 QTL in the genetic background of rice; however, other leaf traits such as porosity and density seemed to be independent of it. Artificial removal of the LGF resulted in partial loss of tolerance, showing increased ethylene production and early induction of anoxia-related genes (SUB1A-1, ACS5, Ramy3D and ADH1) which manifested symptoms such as increased stem elongation, faster chlorophyll and starch breakdown, and partial loss of quiescence in SUB1-containing rice genotypes. Stripping of the LGF resulted in early and enhanced induction of SUB1A-1, indicating a quicker perception of stress. CONCLUSIONS: The presence of SUB1 in the genetic background positively influences surface hydrophobicity and the concomitant LGF thickness of rice. Furthermore, LGF helps in terms of providing better ethylene dissipation and reduced in planta accumulation, owing to the slowing down of ethylene-induced leaf senescence under submergence stress.

Tissue Tolerance Coupled With Ionic Discrimination Can Potentially Minimize the Energy Cost of Salinity Tolerance in Rice.

Salinity is one of the major constraints in rice production. To date, development of salt-tolerant rice cultivar is primarily focused on salt-exclusion strategies, which incur greater energy cost. The present study aimed to evaluate a balancing strategy of ionic discrimination vis-à-vis tissue tolerance, which could potentially minimize the energy cost of salt tolerance in rice. Four rice genotypes, viz., FL478, IR29, Kamini, and AC847, were grown hydroponically and subjected to salt stress equivalent to 12 dS m-1 at early vegetative stage. Different physiological observations (leaf chlorophyll content, chlorophyll fluorescence traits, and tissue Na+ and K+ content) and visual scoring suggested a superior Na+-partitioning strategy operating in FL478. A very low tissue Na+/K+ ratio in the leaves of FL478 after 7 days of stress hinted the existence of selective ion transport mechanism in this genotype. On the contrary, Kamini, an equally salt-tolerant genotype, was found to possess a higher leaf Na+/K+ ratio than does FL478 under similar stress condition. Salt-induced expression of different Na+ and K+ transporters indicated significant upregulation of SOS, HKT, NHX, and HAK groups of transporters in both leaves and roots of FL478, followed by Kamini. The expression of plasma membrane and vacuolar H+ pumps (OsAHA1, OsAHA7, and OsV-ATPase) were also upregulated in these two genotypes. On the other hand, IR29 and AC847 showed greater salt susceptibility owing to excess upward transport of Na+ and eventually died within a few days of stress imposition. But in the "leaf clip" assay, it was found that both IR29 and Kamini had high tissue-tolerance and chlorophyll-retention abilities. On the contrary, FL478, although having higher ionic-discrimination ability, showed the least degree of tissue tolerance as evident from the LC50 score (amount of Na+ required to reduce the initial chlorophyll content to half) of 336 mmol g-1 as against 459 and 424 mmol g-1 for IR29 and Kamini, respectively. Overall, the present study indicated that two components (ionic selectivity and tissue tolerance) of salt tolerance mechanism are distinct in rice. Unique genotypes like Kamini could effectively balance both of these strategies to achieve considerable salt tolerance, perhaps with lesser energy cost.

Ionic selectivity and coordinated transport of Na+ and K+ in flag leaves render differential salt tolerance in rice at the reproductive stage.

MAIN CONCLUSION: The present study shows that salt tolerance in the reproductive stage of rice is primarily governed by the selective Na+ and K+ transport from the root to upper plant parts. Ionic discrimination at the flag leaf, governed by differential expression of Na+- and K+-specific transporters/ion pumps, is associated with reduced spikelet sterility and reproductive stage salt tolerance. Reproductive stage salt tolerance is crucial in rice to guarantee yield under saline condition. In the present study, differential ionic selectivity and the coordinated transport (from root to flag leaf) of Na+ and K+ were investigated to assess their impact on reproductive stage salt tolerance. Four rice genotypes having differential salt sensitivity were subjected to reproductive stage salinity stress in pots. The selective Na+ and K+ transport from the root to upper plant parts was observed in tolerant genotypes. We noticed that prolonged salt exposure did not alter flag leaf greenness even up to 6 weeks; however, it had a detrimental effect on panicle development especially in the salt-susceptible genotype Sabita. But more precise chlorophyll fluorescence imaging analysis revealed salinity-induced damages in Sabita. The salt-tolerant genotype Pokkali (AC41585), a potential Na+ excluder, managed to sequester higher Na+ load in the roots with little upward transport as evident from greater expression of HKT1 and HKT2 transporters. In contrast, the moderately salt-tolerant Lunidhan was less selective in Na+ transport, but possessed a higher capacity to Na+ sequestration in leaves. Higher K+ uptake and tissue-specific redistribution mediated by HAK and AKT transporters showed robust control in selective K+ movement from the root to flag leaf and developing panicles. On the contrary, expressions of Na+-specific transporters in developing panicles were either down-regulated or unaffected in tolerant and moderately tolerant genotypes. Yet, in the panicles of the susceptible genotype Sabita, some of the Na+-specific transporter genes (SOS1, HKT1;5, HKT2;4) were upregulated. Apart from the ionic regulation strategy, cellular energy balance mediated by different plasma-membrane and tonoplastic H+-pumps were also associated with the reproductive stage salt tolerance in rice.

Detection of stable QTLs for grain protein content in rice (Oryza sativa L.) employing high throughput phenotyping and genotyping platforms.

Lack of appropriate donors, non-utilization of high throughput phenotyping and genotyping platforms with high genotype × environment interaction restrained identification of robust QTLs for grain protein content (GPC) in rice. In the present investigation a  BC3F4 mapping population was developed using grain protein donor, ARC10075 and high-yielding cultivar Naveen and 190 lines were genotyped using 40 K Affimetrix custom SNP array with the objective to identify stable QTLs for protein content. Three of the identified QTLs, one for GPC (qGPC1.1) and the other two for single grain protein content (qSGPC2.1, qSGPC7.1) were stable over the environments explaining  13%, 14% and 7.8% of the phenotypic variances, respectively. Stability and repeatability of these additive QTLs were supported by the synergistic additive effects of multi-environmental-QTLs. One epistatic-QTL, independent of  the  main effect QTL was detected over the environment for SGPC. A few functional genes governing seed storage protein were hypothesised inside these identified QTLs. The qGPC1.1 was validated by NIR Spectroscopy-based high throughput phenotyping in BC3F5 population. Higher glutelin content was estimated in high-protein lines with the introgression of qGPC1.1 in telomeric region of short arm of chromosome 1. This was supported by the postulation of probable candidate gene inside this QTL region encoding glutelin family proteins.

Transcriptome profiling uncovers ß-galactosidases of diverse domain classes influencing hypocotyl development in jute (Corchorus capsularis L.).

Enzyme ß-galactosidase (EC 3.2.1.23) is known to influence vascular differentiation during early vegetative growth of plants, but its role in hypocotyl development is not yet fully understood. We generated the hypocotyl transcriptome data of a hypocotyl-defect jute (Corchorus capsularis L.) mutant (52,393 unigenes) and its wild-type (WT) cv. JRC-212 (44,720 unigenes) by paired-end RNA-seq and identified 11 isoforms of ß-galactosidase, using a combination of sequence annotation, domain identification and structural-homology modeling. Phylogenetic analysis classified the jute ß-galactosidases into six subfamilies of glycoside hydrolase-35 family, which are closely related to homologs from Malvaceous species. We also report here the expression of a ß-galactosidase of glycoside hydrolase-2 family that was earlier considered to be absent in higher plants. Comparative analysis of domain structure allowed us to propose a domain-centric evolution of the five classes of plant ß-galactosidases. Further, we observed 1.8-12.2-fold higher expression of nine ß-galactosidase isoforms in the mutant hypocotyl, which was characterized by slower growth, undulated shape and deformed cell wall. In vitro and in vivo ß-galactosidase activities were also higher in the mutant hypocotyl. Phenotypic analysis supported a significant (P ≤ 0.01) positive correlation between enzyme activity and undulated hypocotyl. Taken together, our study identifies the complete set of ß-galactosidases expressed in the jute hypocotyl, and provides compelling evidence that they may be involved in cell wall degradation during hypocotyl development.

Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem.

Rice and Magnaporthe oryzae constitutes an ideal pathosystem for studying host-pathogen interaction in cereals crops. There are two alternative hypotheses, viz. Arms race and Trench warfare, which explain the co-evolutionary dynamics of hosts and pathogens which are under continuous confrontation. Arms race proposes that both R- and Avr- genes of host and pathogen, respectively, undergo positive selection. Alternatively, trench warfare suggests that either R- or Avr- gene in the pathosystem is under balanced selection intending to stabilize the genetic advantage gained over the opposition. Here, we made an attempt to test the above-stated hypotheses in rice-M. oryzae pathosystem at loci of three R-Avr gene pairs, Piz-t-AvrPiz-t, Pi54-AvrPi54 and Pita-AvrPita using allele mining approach. Allele mining is an efficient way to capture allelic variants existing in the population and to study the selective forces imposed on the variants during evolution. Results of nucleotide diversity, neutrality statistics and phylogenetic analyses reveal that Piz-t, Pi54 and AvrPita are diversified and under positive selection at their corresponding loci, while their counterparts, AvrPiz-t, AvrPi54 and Pita are conserved and under balancing selection, in nature. These results imply that rice-M. oryzae populations are engaged in a trench warfare at least at the three R/Avr loci studied. It is a maiden attempt to study the co-evolution of three R-Avr gene pairs in this pathosystem. Knowledge gained from this study will help in understanding the evolutionary dynamics of host-pathogen interaction in a better way and will also aid in developing new durable blast resistant rice varieties in future.

Analysis of Magnaporthe oryzae Genome Reveals a Fungal Effector, Which Is Able to Induce Resistance Response in Transgenic Rice Line Containing Resistance Gene, Pi54.

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with blast resistance gene Pi54 in rice.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.