Plant Science

‘ Candidatus Liberibacter solanacearum’ is an insect-transmitted bacterium associated with several plant diseases. In the Mediterranean Basin, ‘ Ca. L. solanacearum’ haplotype D is vectored by Bactericera trigonica and can severely infect carrot plants leading to abnormal growth phenotypes and significant yield losses. Insecticide applications are insufficient to suppress disease spread and damage, and additional means for disease control are needed. In the current study, we evaluated the resistance of 97 carrot accessions to the bacterial pathogen ‘ Ca. L. solanacearum’ and its associated symptoms. Accessions (Western and Asian types) were first screened in two commercial carrot fields. We found that Western type accessions were less prone to develop disease symptoms in both fields and were less frequently visited by the insect vector in one field. Overall, 22 Asian and five Western accessions with significantly lower disease incidence compared with the commercial cultivar were found. These accessions were then inoculated with ‘ Ca. L. solanacearum’ under controlled conditions and were assessed for disease incidence, insect oviposition, and bacterial relative titer. Five accessions (three Asian and two Western) had significantly lower disease incidence compared with the reference cultivar. Interestingly, disease incidence was not necessarily in line with insect oviposition or in planta bacterial titer, which may indicate that other, perhaps physiological, differences among the accessions may govern the susceptibility of plants to the disease. The resistant accessions found in this study could be used in future resistance breeding programs and to better understand the underlying mechanisms of resistance to ‘ Ca. L. solanacearum’.

Abstract Citrus huanglongbing (HLB), associated with the unculturable phloem‐limited bacterium “ Candidatus Liberibacter asiaticus” ( C Las), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of C Las remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the C Las genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis . The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H 2 O 2 and O 2 − ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri . This study reveals a novel mechanism underlying how C Las uses a prophage‐encoded effector, AGH17488, to target a reactive oxygen species accumulation‐related gene, APX6 , in the host to facilitate its infection.

AbstractIncreasing evidence is proving the biological significance of the phytoplasma-secreted proteins. However, besides a few Sec-dependent secretory proteins, no other phytoplasma-secreted proteins have been reported yet. Candidatus Phytoplasma ziziphi is a phytoplasma that causes witches’-broom, a devastating jujube disease prevalent in east Asia. In this study, using the SecretomeP server coupled with an Escherichia coli-based alkaline phosphatase assay, we identified 25 non-classically secreted proteins (ncSecPs) from Ca. P. ziziphi, a novel type of secreted protein associated with phytoplasmas. Among them, six were characterized as hypersensitive cell death response (HR) suppressors that significantly attenuated both Bax- and INF1-triggered HR and H2O2 accumulation in Nicotiana benthamiana, indicating a so-far unknown role of the phytoplasma-secreted proteins. Further, we demonstrated that despite the diverse subcellular localizations in the N. benthamiana cells, the six HR-suppressing ncSecPs enhanced the gene expression of several known cell death inhibitors, including pathogenesis-related proteins (NbPR-1, NbPR-2, and NbPR-5) and Bax inhibitor-1 (NbBI-1 and NbBI-2). Together, our data indicated that Ca. P. ziziphi has evolved an arsenal of ncSecPs that jointly circumvent HR by activating the plant cell death inhibitors, thus providing new insight into understanding the pathogenesis of phytoplasmas.

Abstract Background Salicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family. Results Here, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators. Conclusion In this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.

Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

Cyanobacterial taxonomy is facing a period of rapid changes thanks to the ease of 16S rRNA gene sequencing and established workflows for description of new taxa. Since the last comprehensive review of the cyanobacterial system in 2014 until 2021, at least 273 species in 140 genera were newly described. These taxa were mainly placed into previously defined orders and families although several new families were proposed. However, the classification of most taxa still relied on hierarchical relationships inherited from the classical morphological taxonomy. Similarly, the obviously polyphyletic orders such as Synechococcales and Oscillatoriales were left unchanged. In this study, the rising number of genomic sequences of cyanobacteria and well‐described reference strains allowed us to reconstruct a robust phylogenomic tree for taxonomic purposes. A less robust but better sampled 16S rRNA gene phylogeny was mapped to the phylogenomic backbone. Based on both these phylogenies, a polyphasic classification throughout the whole phylum of Cyanobacteria was created, with ten new orders and fifteen new families. The proposed system of cyanobacterial orders and families relied on a phylogenomic tree but still employed phenotypic apomorphies where possible to make it useful for professionals in the field. It was, however, confirmed that morphological convergence of phylogenetically distant taxa was a frequent phenomenon in cyanobacteria. Moreover, the limited phylogenetic informativeness of the 16S rRNA gene, resulting in ambiguous phylogenies above the genus level, emphasized the integration of genomic data as a prerequisite for the conclusive taxonomic placement of a vast number of cyanobacterial genera in the future.