Tal2c Activates the Expression of OsF3H04g to Promote Infection as a Redundant TALE of Tal2b in Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae delivers transcription activator-like effectors (TALEs) into plant cells to facilitate infection. Following economic principles, the redundant TALEs are rarely identified in Xanthomonas. Previously, we identified the Tal2b, which activates the expression of the rice 2-oxoglutarate-dependent dioxygenase gene OsF3H03g to promote infection in the highly virulent strain of X. oryzae pv. oryzicola HGA4. Here, we reveal that another clustered TALE, Tal2c, also functioned as a virulence factor to target rice OsF3H04g, a homologue of OsF3H03g. Transferring Tal2c into RS105 induced expression of OsF3H04g to coincide with increased susceptibility in rice. Overexpressing OsF3H04g caused higher susceptibility and less salicylic acid (SA) production compared to wild-type plants. Moreover, CRISPR–Cas9 system-mediated editing of the effector-binding element in the promoters of OsF3H03g or OsF3H04g was found to specifically enhance resistance to Tal2b- or Tal2c-transferring strains, but had no effect on resistance to either RS105 or HGA4. Furthermore, transcriptome analysis revealed that several reported SA-related and defense-related genes commonly altered expression in OsF3H04g overexpression line compared with those identified in OsF3H03g overexpression line. Overall, our results reveal a functional redundancy mechanism of pathogenic virulence in Xoc in which tandem Tal2b and Tal2c specifically target homologues of host genes to interfere with rice immunity by reducing SA.

Overexpression of NDR1 Leads to Pathogen Resistance at Elevated Temperatures

Abiotic and biotic environments influence a myriad of plant-related processes, including growth, development, and the establishment and maintenance of interaction(s) with microbes. As a driver of this signaling between plants and microbes, the role of plant hormones in both surveillance and signaling has emerged as a point of intersection between plant-abiotic and -biotic responses. In the current study, we elucidate a role for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) by exploiting effector-triggered immunity (ETI) to define the regulation of plant host immunity in response to both pathogen infection and elevated temperature. We generated time-series RNA sequencing data of WT Col-0, a NDR1 overexpression line, as well as ndr1 and ics1-2 mutant plants under elevated temperature. Not surprisingly, the NDR1-overexpression line showed genotype-specific gene expression changes related to defense response and immune system function. Interestingly, overexpression of NDR1 revealed a role for NDR1 in immune system function; specifically, we describe a mechanism that intersects with Pseudomonas syringae, type-III effector translocation, R-protein signaling complex stabilization, and sustained levels of SA at elevated temperature during ETI. The results described herein support a role for NDR1 in maintaining cell signaling during simultaneous exposure to elevated temperature and avirulent pathogen stressors.

Persulfidation of Nitrate Reductase 2 Is Involved in L-Cysteine Desulfhydrase-Regulated Rice Drought Tolerance

Hydrogen sulfide (H2S) is an important signaling molecule that regulates diverse cellular signaling pathways through persulfidation. Our previous study revealed that H2S is involved in the improvement of rice drought tolerance. However, the corresponding enzymatic sources of H2S and its regulatory mechanism in response to drought stress are not clear. Here, we cloned and characterized a putative L-cysteine desulfhydrase (LCD) gene in rice, which encodes a protein possessing H2S-producing activity and was named OsLCD1. Overexpression of OsLCD1 results in enhanced H2S production, persulfidation of total soluble protein, and confers rice drought tolerance. Further, we found that nitrate reductase (NR) activity was decreased under drought stress, and the inhibition of NR activity was controlled by endogenous H2S production. Persulfidation of NIA2, an NR isoform responsible for the main NR activity, led to a decrease in total NR activity in rice. Furthermore, drought stress-triggered inhibition of NR activity and persulfidation of NIA2 was intensified in the OsLCD1 overexpression line. Phenotypical and molecular analysis revealed that mutation of NIA2 enhanced rice drought tolerance by activating the expression of genes encoding antioxidant enzymes and ABA-responsive genes. Taken together, our results showed the role of OsLCD1 in modulating H2S production and provided insight into H2S-regulated persulfidation of NIA2 in the control of rice drought stress.

Sweet revenge: AtSWEET12 in plant defense against bacterial pathogens by apoplastic sucrose limitation

Depriving bacterial pathogens of sugars is a potential plant defense strategy. The relevance of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS (SWEETs) in plant susceptibility to pathogens has been established, but their role in plant defense remains unknown. We identified Arabidopsis thaliana SWEETs (AtSWEETs) involved in defense against nonhost and host Pseudomonas syringae pathogens through reverse genetic screening of atsweet1-17 mutants. Double/triple mutant, complementation, and overexpression line analysis, and apoplastic sucrose estimation studies revealed that AtSWEET12 suppresses pathogen multiplication by limiting sucrose availability in the apoplast. Localization studies suggested that plant defense occurred via increased plasma membrane targeting of AtSWEET12 with concomitant AtSWEET11 protein reduction. Moreover, the heterooligomerization of AtSWEET11 and AtSWEET12 was involved in regulating sucrose transport. Our results highlight a PAMP-mediated defense strategy against foliar bacterial pathogens whereby plants control AtSWEET11-mediated sucrose efflux in the apoplast through AtSWEET12. We uncover a fascinating new mechanism of pathogen starvation as a broad-spectrum disease resistance mechanism in parallel with existing immune pathways.

Silicon Modulates Molecular and Physiological Activities in Lsi1 Transgenic and Wild Rice Seedlings under Arsenic Stress

Arsenic is one of the most dangerous metalloids, and silicon is a helpful element supporting plants to withstand stress. In this study, three factors were considered, including rice accessions with three different lines, including Lsi1-RNAi line (LE-R), Lsi1 overexpression line (LE-OE), and their wild type (LE-WT), and silicon and arsenic treatments with two different levels. Analysis of variance in dry weight biomass, protein content, arsenic, and silicon concentration has shown a significant interaction between three factors. Further analysis showed that the silicon concentration of all rice seedlings under silicon treatments increased significantly. The LE-OE line has shown a higher ability to absorb silicon in hydroponic conditions than the wild type, and when the seedlings were exposed to arsenic, the concentration of arsenic in all lines increased significantly. Adding silicon to over-expressed rice lines with the Lsi1 gene creates better arsenic resistance than their wild type. These findings confirmed antagonism between silicon and arsenic, and seedlings exposed to arsenic showed a reduction in silicon concentration in all rice lines. RNA-seq analysis showed 106 differentially expressed genes in the LE-OE line, including 75 up-regulated genes and 31 down-regulated genes. DEGs in the LE-R line were 449 genes, including 190 up-regulated and 259 down-regulated genes. Adding treatment has changed the expression of Calcium-binding EGF domain-containing, Os10g0530500, Os05g0240200 in both LE-OE and LE-R roots. They showed that transgenic cultivars were more resistant to arsenic than wild-type, especially when silicon was added to the culture medium.

Plant DNA methylation is sensitive to parent seed N content and influences the growth of rice

Background Nitrogen (N) is an important nutrient for plant growth, development, and agricultural production. Nitrogen stress could induce epigenetic changes in plants. In our research, overexpression of the OsNAR2.1 line was used as a testing target in rice plants with high nitrogen-use efficiency to study the changes of rice methylation and growth in respond of the endogenous and external nitrogen stress. Results Our results showed that external N deficiency could decrease seed N content and plant growth of the overexpression line. During the filial growth, we found that the low parent seed nitrogen (LPSN) in the overexpression line could lead to a decrease in the filial seed nitrogen content, total plant nitrogen content, yield, and OsNAR2.1 expression (28, 35, 23, and 55%, respectively) compared with high parent seed nitrogen (HPSN) in high nitrogen external supply. However, such decreases were not observed in wild type. Furthermore, methylation sequencing results showed that LPSN caused massive gene methylation changes, which enriched in over 20 GO pathways in the filial overexpression line, and the expression of OsNAR2.1 in LPSN filial overexpression plants was significantly reduced compared to HPSN filial plants in high external N, which was not shown in wild type. Conclusions We suggest that the parent seed nitrogen content decreased induced DNA methylation changes at the epigenetic level and significantly decreased the expression of OsNAR2.1, resulting in a heritable phenotype of N deficiency over two generations of the overexpression line.

RGG1, Involved in the Cytokinin Regulatory Pathway, Controls Grain Size in Rice

Heterotrimeric GTP binding proteins (G proteins) and cytokinin play important roles in regulating plant growth and development. However, little is known about the mechanism by which they coordinate the regulation of grain size in rice. We functionally characterized one gene, RGG1, encoding a type-A Gγ subunit. Strong GUS staining was detected in young panicles and spikelets, suggesting a role for this gene in modulating panicle-related trait development. Overexpression of RGG1 in Nipponbare (NIP) and Wuyunjing 30 (WYJ30) significantly decreased plant height, panicle length and grain length by regulating cell division. However, rgg1 mutants generated by the CRISPR/Cas9 system exhibited no obvious phenotypic differences, which may be due to the extremely low expression level of this gene in vivo. The transcriptomes of young panicles of NIP, the NIP-rgg1–2 mutant and the NIP-OE2 overexpression line were sequenced, and the results showed that many differentially expressed genes (DEGs) were associated with the cytokinin biosynthetic pathway. We confirmed this result by measuring the endogenous cytokinin levels and found that cytokinin content was lower in the overexpression lines. Additionally, increased expression of RGG1 decreased sensitivity to low concentrations of 6-benzylaminopurine (6-BA). Our results reveal a novel G protein—cytokinin module controlling grain size in rice and will be beneficial for understanding the mechanisms by which G proteins regulate grain size and plant development.

GDV1 C-terminal truncation of 39 amino acids disrupts sexual commitment in Plasmodium falciparum

Malaria is a mosquito-borne disease caused by apicomplexan parasites of the genus Plasmodium. Completion of the parasite’s life cycle depends on the transmission of sexual stages, the gametocytes, from an infected human host to the mosquito vector. Sexual commitment occurs in only a small fraction of asexual blood stage parasites and is initiated by external cues. The gametocyte development protein 1 (GDV1) has been described as a key facilitator to trigger sexual commitment. GDV1 interacts with the silencing factor heterochromatin protein 1 (HP1), leading to its dissociation from heterochromatic DNA at the genomic locus encoding AP2-G, the master transcription factor of gametocytogenesis. How this process is regulated is not known. In this study we have addressed the role of protein kinases implicated in gametocyte development. From a pool of available protein kinase KO lines, we identified two kinase knockout lines which fail to produce gametocytes. However, independent genetic verification revealed that both kinases are not required for gametocytogenesis but both lines harbour the same mutation that leads to a truncation in the extreme C-terminus of GDV1. Introduction of the identified nonsense mutation into the genome of wild type parasite lines replicates the observed phenotype. Using a GDV1 overexpression line we show that the truncation in the GDV1 C-terminus does neither interfere with the nuclear import of GDV1 nor its interaction with HP1 in vitro, but appears important to sustain GDV1 protein levels and thereby sexual commitment.ImportanceTransmission of malaria causing Plasmodium species by mosquitos requires the parasite to change from a continuously growing asexual parasite form growing in the blood, to a sexually differentiated form, the gametocyte. Only a small subset of asexual parasites differentiates into gametocytes that are taken up by the mosquito. Transmission represents a bottleneck in the lifecycle of the parasite, so a molecular understanding of the events that lead to stage conversion may identify novel intervention points. Here we screened a subset of kinases we hypothesized to play a role in this process. While we did not identify kinases required for sexual conversion, we identified a mutation in the C-terminus of the Gametocyte Development 1 protein (GDV1), which abrogates sexual development. The mutation destabilises the protein but not its interaction with its cognate binding partner HP1. This suggest an important role for the GDV1 C-terminus beyond trafficking and protein stability.

Functional Analysis of StPHT1;7, a Solanum tuberosum L. Phosphate Transporter Gene, in Growth and Drought Tolerance

PHT1 (phosphate transporter 1) family genes play important roles in regulating plant growth and responding to stress. However, there has been little research on the role of the PHT1 family in potatoes. In this study, using molecular and bioinformatic approaches, 8 PHT1 family genes were identified from the potato genome. StPHT1;7 was highly expressed in the whole potato plants. The overexpression and silence vectors of StPHT1;7 were constructed and transformed into the potato cultivar Desiree. Consequently, StPHT1;7 overexpression (with a relative expression 2–7-fold that in the control) and silence lines (with a relative expression of 0.3%–1% that in the control) were obtained. Their growth vigor was ranked in the order overexpression line > wild type > silence line. In the absence of phosphorus, the root length of the overexpression line was approximately 2.6 times that of the wild type, while the root length of the silence line was approximately 0.6 times that of the wild type. Furthermore, their tolerance to drought stress was ranked as wild type > overexpression line > silence line. These results suggest that StPHT1;7 affects growth and stress tolerance in potato plants.

Ribosomal Protein Large subunit RPL6 modulates salt tolerance in rice

The extra-ribosomal functions of ribosomal proteins RPL6 and RPL23a in stress-responsiveness have emanated from our previous studies on activation tagged mutants of rice screened for water-use efficiency (Moin et al., 2016a). In the present study, we functionally validated the RPL6, a Ribosomal Protein Large subunit member for salt stress tolerance in rice. The overexpression of RPL6 resulted in tolerance to moderate (150 mM) to high (200 mM) levels of salt (NaCl) in rice. The transgenic rice plants expressing RPL6 constitutively showed better phenotypic and physiological responses with high quantum efficiency, accumulation of more chlorophyll and proline contents, and an overall increase in seed yield compared with the wild type in salt stress treatments. An iTRAQ-based comparative proteomic analysis revealed the high expression of about 333 proteins among the 4,378 DEPs in a selected overexpression line of RPL6 treated with 200 mM of NaCl. The functional analysis showed that these highly expressed proteins (HEPs) are involved in photosynthesis, ribosome and chloroplast biogenesis, ion transportation, transcription and translation regulation, phytohormone and secondary metabolite signal transduction. An in silico network analysis of HEPs predicted that RPL6 binds with translation-related proteins and helicases, which coordinately affects the activities of a comprehensive signaling network, thereby inducing tolerance and promoting growth and yield in response to salt stress. Our overall findings identified a novel candidate, RPL6 whose characterization contributed to the existing knowledge on the complexity of salt tolerance mechanism in plants.