Publications

3795

Citrus huanglongbing (HLB), which is caused by the phloem-colonizing bacteria Candidatus Liberibacter asiaticus (CLas), poses a significant threat to citrus production worldwide. The pathogenicity mechanism of HLB remains poorly understood. SEC-dependent effectors (SDEs) have been suggested to play critical roles in the interaction between citrus and CLas. Here, we explored the function of CLIBASIA_05320 (SDE19), a core SDE from CLas, and its interaction with its host target. Our data revealed that SDE19 is expressed at higher level during infection of citrus than that during infection of the Asian citrus psyllid. Subcellular localization assays showed that SDE19 is localized in the nucleus and cytoplasm and is capable of moving from cell to cell in Nicotiana benthamiana. To investigate whether SDE19 facilitates pathogen infection, we generated transgenic Arabidopsis thaliana and citrus plants overexpressing SDE19. Transgenic A. thaliana and citrus plants were more susceptible to Pseudomonas syringae pv. tomato (Pst) and Xanthomonas citri subsp. citri (Xcc), respectively. In addition, RNA-seq analysis demonstrated that overexpression of SDE19 resulted in a reprogramming of expression of genes related to biotic stimulus responses. SDE19 interacts with Citrus sinensis Sec12, a guanine nucleotide exchange factor responsible for the assembly of plant COPII (coat protein II)-coated vesicles, which mediate vesicle trafficking from the ER to the Golgi. SDE19 colocalizes with Sec12 in the ER by binding to its N-terminal catalytic region, affecting the stability of Sec12 through the 26S proteasome. This interaction hinders the secretion of apoplastic defense-related proteins such as PR1, P69B, GmGIP1, and RCR3. Furthermore, the secretion of PR1 and callose deposition is decreased in SDE19-transgenic A. thaliana. Taken together, SDE19 is a novel virulent SDE secreted by CLas that interacts with Sec12 to disrupt vesicle trafficking, inhibit defense-related proteins secretion, and promote bacterial infection. This study sheds light on how CLas manipulates the host vesicle trafficking pathway to suppress the secretion of defense-related proteins and interfere with plant immunity.

‘Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, so far being mainly restricted to Europe. Here, we report draft genome sequences for ‘Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of Prunus persica in Germany and LNS1, leptonecrosis disease of Prunus salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases, assembled into 28 and 16 contigs, respectively, with an average GC content of 21.1% and 20.7%, respectively. Analysis of average nucleotide identity values between genomes further supported the delineation of ‘Ca. Phytoplasma prunorum’ as a species distinct from ‘Ca. Phytoplasma mali’, in agreement with previous phylogenetic data.

In November and December of 2023, samples of cultivars of chrysanthemums (Chrysanthemum morifolium) from a commercial greenhouse facility within the state were submitted to the Oklahoma State University (OSU) Plant Disease and Insect Diagnostic Laboratory (Stillwater, OK). More than 300 symptomatic and asymptomatic stems with flowers and flower buds were submitted. Symptomatic samples were like those reported for aster yellows on multiple plant species, with visible phyllody and virescence. A total of 238 cultivars were processed for phytoplasma detection. Initially, samples were processed in batches based on cultivars. Each batch was made up of 3-9 flowers and/or flower buds from plants belonging to the same cultivar. Genomic DNA was extracted from batched samples using Qiagen DNeasy Plant Mini Kit (Qiagen Inc., MD) following the manufacturer’s instruction. A qPCR assay, using 18S gene as internal control and 23S gene for phytoplasma detection was used to confirm the presence of phytoplasma (Oberhansli et al. 2011). The number of composite samples that tested positive was 67. A nested PCR assay using P1/P7 followed by R16F2n/R16R2 primers (Gundersen and Lee 1996; Smart et al. 1996; Lee et al. 1993; Lee et al. 1998) was used to retest and confirm batched samples that tested positive. PCR products were purified and sequenced at the OSU DNA Core Facility using Sanger Sequencing. A BLAST search of generated sequences on NCBI database confirmed the sequences as aster yellows phytoplasma sequences. To confirm individual plant materials from batches that were positive for aster yellows, DNA extracted from 12 samples representing different cultivars. PCR was carried out using the P1/P7 primers, followed by R16F2n/R16R2 primers as described previously. Another PCR using primer pair L15F1/MapR1 was used to amplify the partial spc operon that includes the complete secY gene (Lee et al. 2010). All PCR products were purified and sequenced at the OSU DNA Core Facility using Sanger Sequencing. BioEdit (https://thalljiscience.github.io/ ) was used to trim and generate consensus sequences of the forward and reverse sequences of all sequenced strains. All sequences have been deposited on the NCBI database (Accession numbers: PP539932-PP539940 and PQ249170-PQ249174 for 16SrRNA and secY genes respectively). The 16S rRNA sequences of strains share between 99.71-99.88% (query coverage= 99-100%, E-value= 0.0) while the secY gene share 98.33-99.49% (query coverage=100%, E-value=0.0) similarity to the 16S rRNA and secY genes of Aster yellows witches'-broom phytoplasma (CP000061.1; Bai et al. 2006). In silico iPhyClassifier 16Sr group/subgroup analyses (Wei et al. 2007; Zhao et al. 2009) showed that these strains belonged to the Candidatus phytoplasma asteris- related strains (subgroup 16SrI-A). Although aster yellows have been reported on other crops in Oklahoma, to our knowledge, this is the first report and confirmation of Candidatus phytoplasma asteris- related strains (subgroup 16SrI-A) causing aster yellows on chrysanthemums in Oklahoma. Considering that this was recovered in one of the major cut flowers growing facilities serving Oklahoma and other states, a concerted effort is required for improved surveillance to mitigate spread of this pathogen.

The symptoms of huanglongbing (HLB), a disease caused by the bacterium Candidatus Liberibacter asiaticus (CLas), are visible on the canopy of citrus plants. They include mottling of leaves followed by dropping and lopsided fruits with premature dropping. Loss in phloem functionality and degradation of the root system are also HLB symptoms with a severe impact on plant growth and production. Some Citrus relatives, such as Poncirus trifoliata and its hybrids, have shown more tolerance to HLB disease and low titers of CLas compared to Citrus species, but little is known about the effects of CLas on their root system. In this study, we investigated the effects of CLas-infected ‘Valencia’ scion on the citrandarin IAC3222 (a hybrid between P. trifoliata and Sunki mandarin) used as rootstock as well as interstock between ‘Valencia’ and Swingle citrumelo rootstock. At 13 months post-inoculation, the cycle threshold values (CT) for CLas in the infected scion samples indicated a high CLas titer (from 15.9 to 22.7) regardless of the rootstock variety or interstock used. However, no CLas-positive samples were detected in the roots of IAC3222 (CT ranging from 37.9 to 40.0), in contrast to all Swingle roots (CT ranging from 27.9 to 31.3). Both root volume and mass were reduced in IAC3222 compared to uninfected ‘Valencia’ scion, suggesting that scion infection damages roots, regardless of whether they are contaminated or not by CLas. The damage to the root system of IAC3222 was significantly less severe than that of the Swingle rootstock. Multivariate hierarchical analysis considering all evaluated parameters clustered the CLas-infected plants grafted on IAC3222 together with the non-inoculated plants. We concluded that the IAC3222 rootstock was less affected by the CLas-infected scion compared to the Swingle rootstock and is a promising rootstock to minimize the HLB effect on plant development.

ABSTRACT‘CandidatusLiberibacter asiaticus’ (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen’ s biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared to previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB.FundingThis work is supported by USDA National Institute of Food and Agriculture award No. 2020-70029-33197 to W.M and A.L.