StMLP1, as a Kunitz trypsin inhibitor, enhances potato resistance and specifically expresses in vascular bundles during Ralstonia solanacearum infection.

Miraculin-like proteins (MLPs), members of the Kunitz trypsin inhibitor (KTI) family that are present in various plants, have been discovered to have a role in defending plants against pathogens. In this study, we identified a gene StMLP1 in potato that belongs to the KTI family. We found that the expression of StMLP1 gradually increases during Ralstonia solanacearum (R. solanacearum) infection. We characterized the promoter of StMLP1 as an inducible promoter that can be triggered by R. solanacearum and as a tissue-specific promoter with specificity for vascular bundle expression. Our findings demonstrate that StMLP1 exhibits trypsin inhibitor activity, and that its signal peptide is essential for proper localization and function. Overexpression of StMLP1 in potato can enhance the resistance to R. solanacearum. Inhibiting the expression of StMLP1 during infection accelerated the infection by R. solanacearum to a certain extent. In addition, the RNA-seq results of the overexpression-StMLP1 lines indicated that StMLP1 was involved in potato immunity. All these findings in our study reveal that StMLP1 functions as a positive regulator that is induced and specifically expressed in vascular bundles in response to R. solanacearum infection.

Protein phosphatase StTOPP6 negatively regulates potato bacterial wilt resistance by modulating MAPK signaling.

Potato (Solanum tuberosum) is an important crop globally and is grown across many regions in China, where it ranks fourth in the list of staple foods. However, its production and quality are severely affected by bacterial wilt caused by Ralstonia solanacearum. In this study, we identified StTOPP6, which belongs to the type one protein phosphatase (TOPP) family, and found that transient knock down of StTOPP6 in potato increased resistance against R. solanacearum. RNA-seq analysis showed that knock down of StTOPP6 activated immune responses, and this defense activation partly depended on the mitogen-activated protein kinase (MAPK) signal pathway. StTOPP6 inhibited the expression of StMAPK3, while overexpression of StMAPK3 enhanced resistance to R. solanacearum, supporting the negative role of StTOPP6 in plant immunity. Consistent with the results of knock down of StTOPP6, overexpressing the phosphatase-dead mutation StTOPP6m also attenuated infection and up-regulated MAPK3, showing that StTOPP6 activity is required for disease. Furthermore, we found that StTOPP6 affected the StMAPK3-mediated downstream defense pathway, eventually suppressing the accumulation of reactive oxygen species (ROS). Consistent with these findings, plants with knock down of StTOPP6, overexpression of StTOPP6m, and overexpression of StMAPK3 all displayed ROS accumulation and enhanced resistance to R. solanacearum. Taken together, the findings of our study demonstrate that StTOPP6 negatively regulates resistance to bacterial wilt by affecting the MAPK3-mediated pathway.

Screening for Resistance Resources against Bacterial Wilt in Wild Potato.

Potato is an important crop, used not only for food production but also for various industrial applications. With the introduction of the potato as a staple food strategy, the potato industry in China has grown rapidly. However, issues related to bacterial wilt, exacerbated by factors such as seed potato transportation and continuous cropping, have become increasingly severe in the primary potato cultivation regions of China, leading to significant economic losses. The extensive genetic diversity of Ralstonia solanacearum (R. solanacearum), which is the pathogen of bacterial wilt, has led to a lack of highly resistant potato genetic resources. There is a need to identify and cultivate potato varieties with enhanced resistance to reduce the adverse impact of this disease on the industry. We screened 55 accessions of nine different wild potato species against the bacterial wilt pathogen R. solanacearum PO2-1, which was isolated from native potato plants and belongs to phylotype II. Three accessions of two species (ACL24-2, PNT880-3, and PNT204-23) were identified with high resistance phenotypes to the tested strains. We found these accessions also showed high resistance to different phylotype strains. Among them, only PNT880-3 was capable of flowering and possessed viable pollen, and it was diploid. Consistent with the high resistance, decreased growth of R. solanacearum was detected in PNT880-3. All these findings in our study reveal that the wild potato PNT880-3 was a valuable resistance source to bacterial wilt with breeding potential.

Comparative genomic analysis of Ralstonia solanacearum reveals candidate avirulence effectors in HA4-1 triggering wild potato immunity.

Ralstonia solanacearum is the causal agent of potato bacterial wilt, a major potato bacterial disease. Among the pathogenicity determinants, the Type III Secretion System Effectors (T3Es) play a vital role in the interaction. Investigating the avirulent T3Es recognized by host resistance proteins is an effective method to uncover the resistance mechanism of potato against R. solanacearum. Two closely related R. solanacearum strains HA4-1 and HZAU091 were found to be avirulent and highly virulent to the wild potato Solanum albicans 28-1, respectively. The complete genome of HZAU091 was sequenced in this study. HZAU091 and HA4-1 shared over 99.9% nucleotide identity with each other. Comparing genomics of closely related strains provides deeper insights into the interaction between hosts and pathogens, especially the mechanism of virulence. The comparison of type III effector repertoires between HA4-1 and HZAU091 uncovered seven distinct effectors. Two predicted effectors RipA5 and the novel effector RipBS in HA4-1 could significantly reduce the virulence of HZAU091 when they were transformed into HZAU091. Furthermore, the pathogenicity assays of mutated strains HA4-1 ΔRipS6, HA4-1 ΔRipO1, HA4-1 ΔRipBS, and HA4-1 ΔHyp6 uncovered that the absence of these T3Es enhanced the HA4-1 virulence to wild potato S. albicans 28-1. This result indicated that these T3Es may be recognized by S. albicans 28-1 as avirulence proteins to trigger the resistance. In summary, this study provides a foundation to unravel the R. solanacearum-potato interaction and facilitates the development of resistance potato against bacterial wilt.

A novel effector RipBT contributes to Ralstonia solanacearum virulence on potato.

Ralstonia solanacearum is one of the most destructive plant-pathogenic bacteria, infecting more than 200 plant species, including potato (Solanum tuberosum) and many other solanaceous crops. R. solanacearum has numerous pathogenicity factors, and type III effectors secreted through type III secretion system (T3SS) are key factors to counteract host immunity. Here, we show that RipBT is a novel T3SS-secreted effector by using a cyaA reporter system. Transient expression of RipBT in Nicotiania benthamiana induced strong cell death in a plasma membrane-localization dependent manner. Notably, mutation of RipBT in R. solanacearum showed attenuated virulence on potato, while RipBT transgenic potato plants exhibited enhanced susceptibility to R. solanacearum. Interestingly, transcriptomic analyses suggest that RipBT may interfere with plant reactive oxygen species (ROS) metabolism during the R. solanacearum infection of potato roots. In addition, the expression of RipBT remarkably suppressed the flg22-induced pathogen-associated molecular pattern-triggered immunity responses, such as the ROS burst. Taken together, RipBT acts as a T3SS effector, promoting R. solanacearum infection on potato and presumably disturbing ROS homeostasis.

Ralstonia solanacearum type III effector RipAS associates with potato type one protein phosphatase StTOPP6 to promote bacterial wilt.

The bacterial pathogen Ralstonia solanacearum (R. solanacearum) delivered type III secretion effectors to inhibit the immune system and cause bacterial wilt on potato. Protein phosphatases are key regulators in plant immunity, which pathogens can manipulate to alter host processes. Here, we show that a type III effector RipAS can reduce the nucleolar accumulation of a type one protein phosphatase (PP1) StTOPP6 to promote bacterial wilt. StTOPP6 was used as bait in the Yeast two-Hybrid (Y2H) assay and acquired an effector RipAS that interacts with it. RipAS was characterized as a virulence effector to contribute to R. solanacearum infection, and stable expression of RipAS in potato impaired plant resistance against R. solanacearum. Overexpression of StTOPP6 showed enhanced disease symptoms when inoculated with wild strain UW551 but not the ripAS deletion mutant, indicating that the expression of StTOPP6 facilitates the virulence of RipAS. RipAS reduced the nucleolar accumulation of StTOPP6, which occurred during R. solanacearum infection. Moreover, the association also widely existed between other PP1s and RipAS. We argue that RipAS is a virulence effector associated with PP1s to promote bacterial wilt.

Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a "species complex" due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong'an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.

A systematic screen of conserved Ralstonia solanacearum effectors reveals the role of RipAB, a nuclear-localized effector that suppresses immune responses in potato.

Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long-term co-evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB-expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild-type potato, transcriptomic analysis of transgenic ripAB-expressing potato plants showed a significant down-regulation of Ca2+ signalling-related genes in the enriched Plant-Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down-regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml-cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune-associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+ -dependent gene expression to promote disease development in potato.