A 'Candidatus Liberibacter solanacearum' Haplotype B-Specific Family of Candidate Bacterial Effectors.

'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.

CLIBASIA_00460 Disrupts Hypersensitive Response and Interacts with Citrus Rad23 Proteins.

'Candidatus Liberibacter asiaticus' (CLas) is a bacterium that causes Huanglongbing, also known as citrus greening, in citrus plants. 'Candidatus Liberibacter solanacearum' (Lso) is a close relative of CLas and in the US it infects solanaceous crops, causing zebra chip disease in potato. Previously, we have identified the Lso hypothetical protein effector 1 (Lso-HPE1). This protein uses a signal peptide for secretion; disrupts programmed cell death; and interacts with tomato RAD23c, d, and e proteins, but not with RAD23a. In this study, we evaluated whether CLIBASIA_00460, the CLas homolog of Lso-HPE1 interacted with citrus RAD23 proteins and disrupted their programmed cell death. Based on the yeast two-hybrid assay results, CLIBASIA_00460 interacted with citrus RAD23c and RAD23d, but not with citrus RAD23b. These results were confirmed using bimolecular fluorescence complementation assays, which showed that these interactions occurred in cell puncta, but not in the nucleus or cytoplasm. Additionally, CLIBASIA_00460 was able to disrupt the PrfD1416V-induced hypersensitive response. Therefore, based on the similar interactions between Lso-HPE1 and CLIBASIA_00460 with the host RAD23 proteins and their ability to inhibit cell death in plants, we propose that these effectors may have similar functions during plant infection.

TaCol-B5 modifies spike architecture and enhances grain yield in wheat.

Spike architecture influences grain yield in wheat. We report the map-based cloning of a gene determining the number of spikelet nodes per spike in common wheat. The cloned gene is named TaCOL-B5 and encodes a CONSTANS-like protein that is orthologous to COL5 in plant species. Constitutive overexpression of the dominant TaCol-B5 allele but without the region encoding B-boxes in a common wheat cultivar increases the number of spikelet nodes per spike and produces more tillers and spikes, thereby enhancing grain yield in transgenic plants under field conditions. Allelic variation in TaCOL-B5 results in amino acid substitutions leading to differential protein phosphorylation by the protein kinase TaK4. The TaCol-B5 allele is present in emmer wheat but is rare in a global collection of modern wheat cultivars.

HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation.

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

Early molecular events associated with nitrogen deficiency in rice seedling roots.

Nitrogen (N) deficiency is one of the most common problems in rice. The symptoms of N deficiency are well documented, but the underlying molecular mechanisms are largely unknown in rice. Here, we studied the early molecular events associated with N starvation (-N, 1 h), focusing on amino acid analysis and identification of -N-regulated genes in rice roots. Interestingly, levels of glutamine rapidly decreased within 15 min of -N treatment, indicating that part of the N-deficient signals could be mediated by glutamine. Transcriptome analysis revealed that genes involved in metabolism, plant hormone signal transduction (e.g. abscisic acid, auxin, and jasmonate), transporter activity, and oxidative stress responses were rapidly regulated by -N. Some of the -N-regulated genes encode transcription factors, protein kinases and protein phosphatases, which may be involved in the regulation of early -N responses in rice roots. Previously, we used similar approaches to identify glutamine-, glutamate-, and ammonium nitrate-responsive genes. Comparisons of the genes induced by different forms of N with the -N-regulated genes identified here have provided a catalog of potential N regulatory genes for further dissection of the N signaling pathwys in rice.

Identification of early ammonium nitrate-responsive genes in rice roots.

Ammonium has long been used as the predominant form of nitrogen source for paddy rice (Oryza sativa). Recently, increasing evidence suggests that nitrate also plays an important role for nitrogen acquisition in the rhizosphere of waterlogged paddy rice. Ammonium and nitrate have a synergistic effect on promoting rice growth. However, the molecular responses induced by simultaneous treatment with ammonium and nitrate have been less studied in rice. Here, we performed transcriptome analysis to identify genes that are rapidly regulated by ammonium nitrate (1.43 mM, 30 min) in rice roots. The combination of ammonium and nitrate preferentially induced the expression of nitrate-responsive genes. Gene ontology enrichment analysis revealed that the early ammonium nitrate-responsive genes were enriched in "regulation of transcription, DNA-dependent" and "protein amino acid phosphorylation" indicating that some of the genes identified in this study may play an important role in nitrogen sensing and signaling. Several defense/stress-responsive genes, including some encoding transcription factors and mitogen-activated protein kinase kinase kinases, were also rapidly induced by ammonium nitrate. These results suggest that nitrogen metabolism, signaling, and defense/stress responses are interconnected. Some of the genes identified here may be involved in the interaction of nitrogen signaling and defense/stress-response pathways in plants.

Exogenous glutamate rapidly induces the expression of genes involved in metabolism and defense responses in rice roots.

BACKGROUND: Glutamate is an active amino acid. In addition to protein synthesis and metabolism, increasing evidence indicates that glutamate may also function as a signaling molecule in plants. Still, little is known about the nutritional role of glutamate and genes that are directly regulated by glutamate in rice. RESULTS: Exogenous glutamate could serve as a nitrogen nutrient to support the growth of rice seedlings, but it was not as effective as ammonium nitrate or glutamine. In nitrogen-starved rice seedlings, glutamate was the most abundant free amino acid and feeding of glutamate rapidly and significantly increased the endogenous levels of glutamine, but not glutamate. These results indicated that glutamate was quickly metabolized and converted to the other nitrogen-containing compounds in rice. Transcriptome analysis revealed that at least 122 genes involved in metabolism, transport, signal transduction, and stress responses in the roots were rapidly induced by 2.5 mM glutamate within 30 min. Many of these genes were also up-regulated by glutamine and ammonium nitrate. Still, we were able to identify some transcription factor, kinase/phosphatase, and elicitor-responsive genes that were specifically or preferentially induced by glutamate. CONCLUSIONS: Glutamate is a functional amino acid that plays important roles in plant nutrition, metabolism, and signal transduction. The rapid and specific induction of transcription factor, kinase/phosphatase and elicitor-responsive genes suggests that glutamate may efficiently amplify its signal and interact with other signaling pathways to regulate metabolism, growth and defense responses in rice.

Gene expression profiling of rice seedlings in response to glutamine treatment.

Glutamine, the most abundant free amino acid in humans (Curi et al., 2007 [1]), has many functions. In addition to protein, amino acid, and nucleic acid biosynthesis, glutamine also regulates the expression of genes related to metabolism, cell defense, and signal transduction in humans (Curi et al., 2007 [1]; Brasse-Lagnel et al., 2009 [2]). Glutamine is also one of the major forms of nitrogen in rice (Fukumorita and Chino, 1982 [3]). In addition to metabolic and nutritional effects, glutamine may function as a signaling molecule to regulate gene expression in plants. To this end, we used microarray analysis to identify genes that are rapidly induced by 2.5 mM glutamine in rice roots. The results revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min (Kan et al., 2015 [4]). Here, we provide the details of the experimental procedure associated with our microarray data deposited in NCBI's Gene Expression Omnibus (GEO ID: GSE56770).

Glutamine rapidly induces the expression of key transcription factor genes involved in nitrogen and stress responses in rice roots.

BACKGROUND: Glutamine is a major amino donor for the synthesis of amino acids, nucleotides, and other nitrogen-containing compounds in all organisms. In addition to its role in nutrition and metabolism, glutamine can also function as a signaling molecule in bacteria, yeast, and humans. By contrast, the functions of glutamine in nutrition and as a signaling molecule remain unclear in plants. RESULTS: We demonstrated that glutamine could effectively support the growth of rice seedlings. In glutamine-treated rice roots, the glutamine contents increased dramatically, whereas levels of glutamate remained relatively constant. Transcriptome analysis of rice roots revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min. Interestingly, 10 of the 35 early glutamine responsive genes encode putative transcription factors, including two LBD37-like genes that are involved in the regulation of nitrogen metabolism. Glutamine also rapidly induced the expression of the DREB1A, IRO2, and NAC5 transcription factor genes, which are involved in the regulation of stress responses. CONCLUSIONS: In addition to its role as a metabolic fuel, glutamine may also function as a signaling molecule to regulate gene expression in plants. The rapid induction of transcription factor genes suggests that glutamine may efficiently amplify its signal and interact with the other signal transduction pathways to regulate plant growth and stress responses. Thus, glutamine is a functional amino acid that plays important roles in plant nutrition and signal transduction.