Huanglongbing (HLB) is a devastating disease of citrus, caused by the phloem-colonizing bacteriumCandidatusLiberibacter asiaticus (CLas). Here, we present evidence that HLB is an immune-mediated disease. We show that CLas infection ofCitrus sinensisstimulates systemic and chronic immune responses in phloem tissue, including callose deposition, production of reactive oxygen species (ROS) such as H₂O₂, and induction of immunity-related genes. The infection also upregulates genes encoding ROS-producing NADPH oxidases, and downregulates antioxidant enzyme genes, supporting that CLas causes oxidative stress. CLas-triggered ROS production localizes in phloem-enriched bark tissue and is followed by systemic cell death of companion and sieve element cells. Inhibition of ROS levels in CLas-positive stems by NADPH oxidase inhibitor diphenyleneiodonium (DPI) indicates that NADPH oxidases contribute to CLas-triggered ROS production. To investigate potential treatments, we show that addition of the growth hormone gibberellin (known to have immunoregulatory activities) upregulates genes encoding H₂O₂-scavenging enzymes and downregulates NADPH oxidases. Furthermore, foliar spray of HLB-affected citrus with gibberellin or antioxidants (uric acid, rutin) reduces H₂O₂concentrations and cell death in phloem tissues and reduces HLB symptoms. Thus, our results indicate that HLB is an immune-mediated disease that can be mitigated with antioxidants and gibberellin.

Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant immunity induced by mobile signals produced in the local leaves where the initial infection occurs. Although multiple structurally unrelated signals have been proposed, the mechanisms responsible for perception of these signals in the systemic leaves are unknown. Here, we show that exogenously applied nicotinamide adenine dinucleotide (NAD⁺) moves systemically and induces systemic immunity. We demonstrate that the lectin receptor kinase (LecRK), LecRK-VI.2, is a potential receptor for extracellular NAD⁺(eNAD⁺) and NAD⁺phosphate (eNADP⁺) and plays a central role in biological induction of SAR. LecRK-VI.2 constitutively associates with BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) in vivo. Furthermore, BAK1 and its homolog BAK1-LIKE1 are required for eNAD(P)⁺signaling and SAR, and the kinase activities of LecR-VI.2 and BAK1 are indispensable to their function in SAR. Our results indicate that eNAD⁺is a putative mobile signal, which triggers SAR through its receptor complex LecRK-VI.2/BAK1 inArabidopsis thaliana.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus.

Zoysia matrella, widely used in lawns and sports fields, is of great economic and ecological value. Z. matrella is an allotetraploid species (2n = 4x = 40) in the genus zoysia under the subfamily Chloridoideae. Despite its ecological impacts and economic importance, the subfamily Chloridoideae has received little attention in genomics studies. As a result, limited genetic and genomic information are available for this subfamily, which have impeded progress in understanding evolutionary history of grasses in this important lineage. The lack of a high-resolution genetic map has hampered efforts to improve zoysiagrass using molecular genetic tools. We used restriction site-associated DNA sequencing (RADSeq) approach and a segregating population developed from the cross between Z. matrella cultivars ‘Diamond’ and ‘Cavalier’ to construct high-resolution genetic maps of Z. matrella. The genetic map of Diamond consists of 2,375 Single Nucleotide Polymorphism (SNP) markers mapped on 20 linkage groups (LGs) with a total length of 1754.48 cM and an average distance between adjacent markers at 0.74 cM. The genetic map of Cavalier contains 3,563 SNP markers on 20 LGs, covering 1824.92 cM, with an average distance between adjacent markers at 0.51 cM. A higher level of genome collinearity between Z. matrella and rice than that between Z. matrella and sorghum was revealed by comparative genomic analysis. Pairwise comparison revealed that two independent nested chromosome fusion events occurred after Z. matrella and sorghum split from a common ancestor. The high-resolution linkage maps were applied into mapping QTLs associated with fall armyworm (FAW) resistance and six loci located on LGs 8 and 20 were detected to be significantly associated with FAW resistance. The high-resolution linkage maps provide anchor points for comparative genomics analysis between Z. matrella and other grass species. Our comparative genomic analysis suggested that the chromosome number reduction from 12 to 10 had occurred independently via a single-step in the subfamilies Chloridoideae and Panicoideae. The high-resolution genetic maps provide an essential framework for mapping QTLs associated with economically and agronomically important traits. The major QTLs mapped on LG8 of the Cavalier map provide a starting point for cloning FAW resistance genes and further studies for a better understanding of FAW resistance in zoysiagrass.